Methods and Means for Obtaining Modified Phenotypes

ABSTRACT

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprises simultaneously both sense and antisense nucleotide sequence.

This application is a continuation of U.S. application Ser. No.11/364,183, filed Mar. 1, 2006, and issuing as U.S. Pat. No. 9,441,239,which is a continuation of U.S. application Ser. No. 10/755,328, filedJan. 13, 2004 and is a continuation of U.S. application Ser. No.09/287,632, filed Apr. 7, 1999, which issued as U.S. Pat. No. 8,598,332,and which claims the benefit of Provisional Application No. 60/198,254,filed Aug. 3, 1998 and Provisional Application No. 60/198,240, filedApr. 8, 1998. The contents of each of these applications are herebyincorporated by reference in their entirety for all purposes.

1. FIELD OF THE INVENTION

The invention relates to methods for reducing the phenotypic expressionof a nucleic acid sequence of interest in eucaryotic cells, particularlyplant cells, by simultaneously providing the cells with chimeric genesencoding sense and anti sense RNA molecules comprising nucleotidesequences respectively homologous and complementary to at least part ofthe nucleotide sequence of the nucleic acid of interest. The sense andantisense RNA molecules may be provided as one RNA molecule, wherein thesense and antisense RNA may be linked by a spacer nucleotide sequenceand are capable of forming a double stranded RNA molecule. In one aspectof the invention, the methods are directed towards reducing viralinfection, resulting in extreme virus resistance. In another embodimentthe methods are directed towards reducing the phenotypic expression ofan endogenous gene in a plant cell. The invention further relates tohigh throughput screening methods for identifying the phenotype endowedby the nucleic acid of interest in plant cells. Also provided are plantcells comprising such RNA molecules, as well as plants consistingessentially of such plant cells.

2. BACKGROUND OF THE INVENTION

In 1985, Sanford and Johnston proposed the concept of parasite-derivedresistance. They postulated that key gene products from a parasiteexpressed in the host in a dysfunctional form, in excess or at a wrongdevelopmental stage, should disrupt the function of the parasite withminimal effect on the host (Sanford & Johnston, 1985). Using the QBbacteriophage as a model, they proposed that expression, in bacteria, ofthe bacteriophage coat protein or modified replicase or an antisense RNAcomplementary to the QB genome could all give resistance. They alsoproposed that such approaches would be applicable, in plants, to plantviruses and particularly the use of a modified plant virus replicase.The expression of the coat protein of the plant virus, tobacco mosaicvirus (TMV), in tobacco was the first practical validation of thisconcept for plant virus resistance. This work (Powell-Abel et al., 1986)showed that the expression of the TMV coat protein, from a transgeneunder the control of the cauliflower mosaic virus 35S promoter,conferred on the plants resistance to TMV. The same group (Powell etal., 1990) showed that, generally, plants expressing higher levels ofcoat protein were more resistant to TMV than plants expressing lowlevels. Since this demonstration there have been very many examples ofplants transformed with virus coat protein genes showing resistance(Table 1). There have also been a number of reports of plant virusresistance in plants expressing wild-type replicase (Braun and Hemenway,1992, Brederode et al., 1995), truncated replicase (Carr et al. 1992),modified replicase (Longstaff et al. 1993), or antisense viral RNA(Kawchuck et al. 1991).

In 1992, Dougherty and colleagues were using different forms of the coatprotein gene of tobacco etch virus (TEV) and discovered that some plantscontaining untranslatable “sense” coat protein genes and antisense coatprotein genes showed extreme resistance (ER) to the virus (Lindbo &Dougherty, 1992 a,b). This resistance was functional at the whole plantlevel and at the single cell level. TEV was unable to replicate inprotoplasts derived from ER plants but replicated to high levels inprotoplasts from non-transgenic tobacco. Dougherty et al. concluded thatthe mechanism creating the extreme resistance for the untranslatablesense construct was not the same as the often reported coatprotein-mediated strategy. They proposed that the mRNA of theuntranslatable sense construct was hybridizing with the minus sensegenome of the virus and interfering with the procession of replicationcomplexes on the minus strand. They suggested that the use of viralsequence that could form intramolecular pairing should be avoided asthis would interfere with their ability to hybridize to the target viralRNA.

TABLE 1 Plant species that have been genetically engineered for virusresistance (from Rebecca Grumet, Hort Science 30[3] 1995) SpeciesViruses Tobacco AIMV, ArMV, CMV, PVX, PVY, (Nicotiana tabacum L.) TEV,TGMV, TMV, TRV, TSV, TSWV Other Nicotiana spp. ACMV, BYMV, CyMV, CyRSV,(N. debneyii, N. benthamiana, BCTV, PEBV, PPV, PVS, N. clevelandii) WMVPotato (Solanum tuberusom L.) PI, RV, PVY Tomato AIMV, CMV, TMV, TYLCV(Lycopersicon esculentum L.) Cucumber (Cucumis sativus L.) CMV Melon(Cucumis melo L.) CMV, ZYMV Alfalfa (Medicago sativa L.) AIMV Papaya(Carica papaya L.) PRSV Corn (Zea mays L.) MDMV Rice (Oryza sativa L.)RSV Rapeseed (Brassica napus L.) TYMV

The Dougherty group expanded their investigations to plants containinguntranslatable sense potato virus Y (PVY) coat protein genes. Theyobtained results similar to those with TEV and showed that the plantswith ER had high transgene copy number, highly active transcription ofthe transgenes, and low levels of steady state mRNA from the PVYtransgene (Lindbo et al. 1993, Smith et al. 1994). The following modelfor this mechanism of the resistance was proposed: the high level oftranscription of the viral transgene triggers a cytoplasmic based, posttranscriptional cellular surveillance system that targets specific RNAsfor elimination. As the transgene encodes a transcript comprising viralsequences the mechanism not only degrades the transgene mRNA but alsothe same sequences in the viral genomic RNA. A key point in this modelis the need for a high level of transcription of the transgene providedby high copy number (3-8; Goodwin et al. 1996). Alternatively, the RNAthreshold required to trigger the mechanism can be reached by a moremodest transcription level aided by the viral RNA from replication inearly infection. This gives rise to a “recovery phenotype” where theplant is initially infected and shows symptoms but then produces newgrowth without virus symptoms and which are extremely resistant toinfection.

This proposal was substantiated by the findings that gene silencing ofnon-viral transgenes could also be due to a post transcriptionalmechanism (Ingelbrecht et al. 1994; de Carvalho Niebel et al. 1995)operating at an RNA level.

A link between non-viral gene silencing and this pathogen derivedresistance was provided by inoculating transgenic plants, in which a GUStransgene was known to be silenced by a post transcriptional mechanism,with a virus containing GUS sequences (English et al. 1996). In thissituation the plants were extremely resistant to the viral infection.However, the same plants were susceptible to the virus if they containedno GUS sequences.

The degree of viral resistance is not always directly related to thelevel of viral transgene transcription (Mueller et al. 1995; English etal. 1996) suggesting that there may be an alternative mechanism ofinducing the resistance. To accommodate these discrepancies, analternative model has been proposed in which the crucial factoraffecting the resistance is not the level but the quality of thetransgene mRNA (English et al. 1996). According to this model, thetransgene can only induce resistance (or gene silencing) if it istranscribed to produce “aberrant” RNA. There have been many examples ofpost-transcriptional gene silencing and methylation of the transgene(Hobbs et al. 1990; Ingelbrecht et al. 1994) and methylation of thetransgene has also been found to be associated in some cases of extremeviral resistance (Smith et al. 1994, English 1996). In the proposedmodel, methylation of the transgene leads to the production of aberrantRNAs which induce the cytoplasmic RNA surveillance system. Baulcombe andEnglish have suggested that this method of induction may be the same asthat found for the silencing of met2 in A. immersus. In this systemtranscription of the met2 RNA was terminated in the methylated regionsof the endogenous gene thus producing aberrant truncated RNAs. It wassuggested that the methylation was a consequence of ectopic interactionbetween the transgene and a homologous region of a corresponding regionof the endogenous gene (Barry et al. 1993). The presence of multipletransgenes would create an increased likelihood of ectopic pairing andis therefore consistent with the correlation between high copy numberand extreme viral resistance (Mueller et al., 1995; Goodwin et al. 1996;Pang et al., 1996).

This whole area has been reviewed recently (e.g. Baulcombe (1996) andStam et al. (1997)) and several models were presented. All models callfor a high degree of sequence specificity because the resistance is very(strain) specific and therefore invoke base pairing interaction with anRNA produced from the transgene. One explanation for the induction ofthe virus resistance or gene silencing with sense transgenes is that theplant's RNA dependent RNA polymerase generates complementary RNAs usingthe transgene mRNA as a template (Schiebel et al. 1993a,b). Thishypothetical complementary RNA (cRNA) has not been detected (Baulcombe1996) but it is expected that the cRNAs will be small and heterodisperseRNAs rather than full complements (Schiebel 1993ab, Baulcombe 1996) andtherefore difficult to detect.

The possible methods of action of the cRNA in mediating the virusresistance or gene silencing (as proposed by Baulcombe, 1996) are:

1: The cRNA hybridizes with transgene mRNA or viral RNA and the duplexbecomes a target for dsRNases;2: The cRNA hybridizes with target RNA to form a duplex which can arresttranslation and consequently have an indirect effect on stability(Green, 1993);3: The duplex formed between the cRNA and viral RNA causes hybrid arrestof translation of co-factors required for viral replication; and4. The hybridization of the cRNA affects intra-molecular base pairingrequired for viral replication.

The current models for virus resistance or gene silencing thus involvethe induction of a cytoplasmic surveillance system by either high levelsof transgene transcription or by the production of aberrant singlestranded mRNA. Once the system is triggered, RNA dependent RNApolymerase makes cRNA from the transgene mRNA. These cRNAs hybridize tothe target RNA either directly affecting its translatability orstability, or marking the RNA for degradation.

U.S. Pat. No. 5,190,131 and EP 0 467 349 A1 describe methods and meansto regulate or inhibit gene expression in a cell by incorporating intoor associating with the genetic material of the cell a non-nativenucleic acid sequence which is transcribed to produce an mRNA which iscomplementary to and capable of binding to the mRNA produced by thegenetic material of that cell.

EP 0 223 399 A1 describes methods to effect useful somatic changes inplants by causing the transcription in the plant cells of negative RNAstrands which are substantially complementary to a target RNA strand.The target RNA strand can be a mRNA transcript created in geneexpression, a viral RNA, or other RNA present in the plant cells. Thenegative RNA strand is complementary to at least a portion of the targetRNA strand to inhibit its activity in vivo.

EP 0 240 208 describes a method to regulate expression of genes encodedfor in plant cell genomes, achieved by integration of a gene under thetranscriptional control of a promoter which is functional in the hostand in which the transcribed strand of DNA is complementary to thestrand of DNA that is transcribed from the endogenous gene(s) one wishesto regulate.

EP 0 647 715 A1 and U.S. Pat. Nos. 5,034,323, 5,231,020 and 5,283,184describe methods and means for producing plants exhibiting desiredphenotypic traits, by selecting transgenotes that comprise a DNA segmentoperably linked to a promoter, wherein transcription products of thesegment are substantially homologous to corresponding transcripts ofendogenous genes, particularly endogenous flavonoid biosynthetic pathwaygenes.

WO 93/23551 describes methods and means for the inhibition of two ormore target genes, which comprise introducing into the plant a singlecontrol gene which has distinct DNA regions homologous to each of thetarget genes and a promoter operative in plants adapted to transcribefrom such distinct regions RNA that inhibits expression of each of thetarget genes.

WO92/13070 describes a method for the regulation of nucleic acidtranslation, featuring a responsive RNA molecule which encodes apolypeptide and further includes a regulatory domain, a substrate regionand a ribosome recognition sequence. This responsive RNA molecule has aninhibitor region in the regulatory domain, which regulatory domain iscomplementary to both a substrate region of the responsive RNA moleculeand to an anti-inhibitor region of a signal nucleic acid such that, inthe absence of the signal nucleic acid, the inhibitor and substrateregions form a base-paired domain the formation of which reduced thelevel of translation of one of the protein-coding regions in theresponsive RNA molecule compared to the level of translation of that oneprotein-coding region observed in the presence of the signal nucleicacid.

Metzlaff et al., 1997 describe a model for the RNA-mediated RNAdegradation and chalcone synthase A silencing in Petunia, involvingcycles of RNA-RNA pairing between complementary sequences followed byendonucleolytic RNA cleavages to describe how RNA degradation is likelyto be promoted. Fire et al., 1998 describe specific genetic interferenceby experimental introduction of double-stranded RNA in Caenorhabditiselegans. The importance of these findings for functional genomics isdiscussed (Wagner and Sun, 1998).

Que et al., 1998 describe distinct patterns of pigment suppression whichare produced by allelic sense and antisense chalcone synthase transgenesin petunia flowers and have also analyzed flower color patterns inplants heterozygous for sense and antisense chalcone synthase alleles.

WO 98/05770 discloses antisense RNA with special secondary structureswhich may be used to inhibit gene expression.

WO 94/18337 discloses transformed plants which have increased ordecreased linolenic acids as well as plants which express a linoleicacid desaturase.

U.S. Pat. No. 5,850,026 discloses an endogenous oil from Brassica seedsthat contains, after crushing and extracting, greater than 86% oleicacid and less than 2.5% α-linolenic acid. The oil also contains lessthan 7% linoleic acid. The Brassica seeds are produced by plants thatcontain seed-specific inhibition of microsomal oleate desaturase andmicrosomal linoleate desaturase gene expression, wherein the inhibitioncan be created by cosuppression or antisense technology.

U.S. Pat. No. 5,638,637 discloses vegetable oil from rapeseeds andrapeseed producing the same, the vegetable oil having an unusually higholeic acid content of 80% to 90% by weight based on total fatty acidcontent.

SUMMARY OF THE INVENTION

The present invention provides methods for reducing the phenotypicexpression of a nucleic acid of interest, which is normally capable ofbeing expressed in a eucaryotic cell, particularly for reducing thephenotypic expression of a gene, particularly a endogenous gene or aforeign transgene, integrated in the genome of a eucaryotic cell or forreducing the phenotypic expression of nucleic acid of interest which iscomprised in the genome of an infecting virus, comprising the step ofintroducing, preferably integrating, in the nuclear genome of theeucaryotic cell, a chimeric DNA comprising a promoter, capable of beingexpressed in that eucaryotic cell, and optionally a DNA region involvedin transcription termination and polyadenylation and in between a DNAregion, which when transcribed, yields an RNA molecule with a nucleotidesequence comprising a sense nucleotide sequence of at least 10consecutive nucleotides, particularly at least about 550 consecutivenucleotides, having between 75 and 100% sequence identity with at leastpart of the nucleotide sequence of the nucleic acid of interest, and anantisense nucleotide sequence including at least 10 consecutivenucleotides, having between about 75% to about 100% sequence identitywith the 10 nucleotide stretch of the complement of the sense nucleotidesequence, wherein the RNA is capable of forming an artificial hairpinRNA structure with a double stranded RNA stem by base-pairing betweenthe regions with sense and antisense nucleotide sequence such that atleast the 10 consecutive nucleotides of the sense sequence base pairwith the 10 consecutive nucleotides of the antisense sequence, resultingin, preferably an artificial hairpin structure. Preferably the chimericDNA is stably integrated in the genome of the DNA.

The invention also provides a method for reducing the phenotypicexpression of a nucleic acid of interest, which is normally capable ofbeing expressed in a eucaryotic cell comprising the step of introducinga chimeric RNA molecule with a nucleotide sequence comprising a sensenucleotide sequence of at least 10 consecutive nucleotides havingbetween 75 and 100% sequence identity with at least part of thenucleotide sequence of the nucleic acid of interest; and an antisensenucleotide sequence including at least 10 consecutive nucleotides,having between about 75% to about 100% sequence identity with 10 ntstretch of the complement of the sense nucleotide sequence; wherein theRNA is capable of forming a double stranded RNA region by base-pairingbetween the regions with sense and antisense nucleotide sequence suchthat at least the 10 consecutive nucleotides of the sense sequence basepair with the 10 consecutive nucleotides of the antisense sequence,resulting in a(n artificial) hairpin RNA structure.

The invention further provides a method for reducing the gene expressionof a gene of interest in plant cells, comprising the step of introducinga first and second chimeric DNA, linked on one recombinant DNA such thatboth chimeric DNAs are integrated together in the nuclear genome of thetransgenic plant cells; wherein the first chimeric DNA comprises aplant-expressible promoter, a first DNA region capable of beingtranscribed into a sense RNA molecule with a nucleotide sequencecomprising a sense nucleotide sequence of at least 10 consecutivenucleotides having between 75 and 100% sequence identity with at leastpart of the nucleotide sequence of the gene of interest and optionally aDNA region involved in transcription termination and polyadenylationfunctioning in plant cells. The second chimeric DNA comprises aplant-expressible promoter, a second DNA region capable of beingtranscribed into an antisense RNA molecule with a nucleotide sequencecomprising an antisense nucleotide sequence including at least 10consecutive nucleotides, having between about 75% to about 100% sequenceidentity with the complement of the at least 10 consecutive nucleotidesof the sense nucleotide sequence and optionally a DNA region involved intranscription termination and polyadenylation functioning in plantcells. The sense and antisense RNA molecules are capable of forming adouble stranded, duplex RNA by base-pairing between the regions whichare complementary.

Also provided by the invention is a method for obtaining virus resistantorganisms, particularly plants, comprising the steps of providing cellsof the organism with a first and second chimeric DNA, wherein the firstchimeric DNA comprises a promoter, a first DNA region capable of beingtranscribed into a sense RNA molecule with a nucleotide sequencecomprising a sense nucleotide sequence of at least 10 consecutivenucleotides having between 75 and 100% sequence identity with at leastpart of the nucleotide sequence of the genome of a virus, capable ofinfecting the plant and a DNA region involved in transcriptiontermination and polyadenylation functioning in plant cells. The secondchimeric DNA comprises a promoter, a second DNA region capable of beingtranscribed into an antisense RNA molecule with a nucleotide sequencecomprising an antisense nucleotide sequence including at least 10consecutive nucleotides, having between about 75% to about 100% sequenceidentity with the complement of at least 10 consecutive nucleotides ofthe sense nucleotide sequence and a DNA region involved in transcriptiontermination and polyadenylation functioning in plant cells. The senseand antisense RNA molecules are capable of forming a double stranded RNAregion by base-pairing between the regions which are complementary.

The first and second chimeric DNA are either integrated separately inthe nuclear genome of the transformed plant cell or they are linked onone recombinant DNA such that both chimeric DNAs are integrated togetherin the nuclear genome of the transgenic plant cells.

The invention also provides a method for identifying a phenotypeassociated with the expression of a nucleic acid of interest in aeucaryotic cell, comprising the steps of selecting within the nucleotidesequence of interest, a target sequence of at least 10 consecutivenucleotides; designing a sense nucleotide sequence corresponding to thelength of the selected target sequence and which has a sequence identityof at least about 75% to about 100% with the selected target sequence,designing an antisense nucleotide sequence which has a sequence identityof at least about 75% to about 100% with the complement of the sensenucleotide sequence and comprises a stretch of at least about 10consecutive nucleotides with 100% sequence identity to the complement ofa part of the sense nucleotide sequence. The method further comprisesthe steps of introducing an RNA molecule comprising both the designedsense and antisense nucleotide sequences into a suitable eucaryotic hostcell comprising the nucleic acid including the nucleotide sequence withhitherto unidentified phenotype; and observing the phenotype by asuitable method.

The invention also provides a eucaryotic cell, comprising a nucleic acidof interest which is normally capable of being phenotypically expressed,further comprising a chimeric DNA molecule comprising a promoter,capable of being expressed in that eucaryotic cell, a DNA region, whichwhen transcribed, yields an RNA molecule with a nucleotide sequencecomprising a sense nucleotide sequence of at least 10 consecutivenucleotides having between 75 and 100% sequence identity with at leastpart of the nucleotide sequence of the nucleic acid of interest and anantisense nucleotide sequence including at least 10 consecutivenucleotides, having between about 75% to about 100% sequence identitywith the complement of the at least 10 consecutive nucleotides of thesense nucleotide sequence wherein the RNA molecule is capable of forminga double stranded RNA region by base-pairing between the regions withsense and antisense nucleotide sequence such that at least said 10consecutive nucleotides of the sense sequence base pair with said 10consecutive nucleotides of the antisense sequence, resulting in ahairpin RNA structure, preferably an artificial hairpin structure and aDNA region involved in transcription termination and polyadenylation,wherein the phenotypic expression of the nucleic acid of interest issignificantly reduced.

Also provided by the invention is a eucaryotic cell, comprising anucleic acid of interest, which is normally capable of beingphenotypically expressed, further comprising a chimeric RNA moleculewith a nucleotide sequence comprising a sense nucleotide sequence of atleast 10 consecutive nucleotides having between 75 and 100% sequenceidentity with at least part of the nucleotide sequence of the nucleicacid of interest and an antisense nucleotide sequence including at least10 consecutive nucleotides, having between about 75% to about 100%sequence identity with the complement of the at least 10 consecutivenucleotides of the sense nucleotide sequence wherein the RNA is capableof forming a double stranded RNA region by base-pairing between theregions with sense and antisense nucleotide sequence such that at leastsaid 10 consecutive nucleotides of the sense sequence basepair with said10 consecutive nucleotides of the antisense sequence, resulting in anartificial hairpin RNA structure.

It is another objective of the invention to provide a eucaryotic cell,comprising a gene of interest, which is normally capable of beingphenotypically expressed, further comprising a first and second chimericDNA, linked on one recombinant DNA such that both chimeric DNAs areintegrated together in the nuclear genome of that eucaryotic cellwherein the first chimeric DNA comprises the following operably linkedparts a promoter capable of being expressed in the eucaryotic cell afirst DNA region capable of being transcribed into a sense RNA moleculewith a nucleotide sequence comprising a sense nucleotide sequence of atleast 10 consecutive nucleotides having between 75 and 100% sequenceidentity with at least part of the nucleotide sequence of the gene ofinterest; and a DNA region involved in transcription termination andpolyadenylation; and wherein the second chimeric DNA comprises thefollowing operably linked parts: a promoter operative in the eucaryoticcell; a second DNA region capable of being transcribed into an antisenseRNA molecule with a nucleotide sequence comprising an antisensenucleotide sequence including at least 10 consecutive nucleotides,having between about 75% to about 100% sequence identity with thecomplement of the at least 10 consecutive nucleotides of the sensenucleotide sequence; a DNA region involved in transcription terminationand polyadenylation; wherein the sense and antisense RNA molecules arecapable of forming a double stranded RNA region by base-pairing betweenthe regions which are complementary.

It is yet another objective of the invention to provide a virusresistant plant, comprising a first and second chimeric DNA integratedin the nuclear genome of its cells, wherein the first chimeric DNAcomprises a plant-expressible promoter, a first DNA region capable ofbeing transcribed into a sense RNA molecule with a nucleotide sequencecomprising a sense nucleotide sequence of at least 10 consecutivenucleotides having between 75 and 100% sequence identity with at leastpart of the nucleotide sequence of the genome of a virus, capable ofinfecting the plant, and a DNA region involved in transcriptiontermination and polyadenylation functioning in plant cells. The secondchimeric DNA comprises a plant-expressible promoter, a second DNA regioncapable of being transcribed into an antisense RNA molecule with anucleotide sequence comprising an antisense nucleotide sequenceincluding at least 10 consecutive nucleotides, having between about 75%to about 100% sequence identity with the complement of the at least 10consecutive nucleotides of the sense nucleotide sequence, and a DNAregion involved in transcription termination and polyadenylationfunctioning in plant cells. The sense and antisense RNA molecules arecapable of forming a double stranded RNA region by base-pairing betweenthe regions which are complementary. The first and second chimeric DNAare integrated either in one locus or in different loci in the nucleargenome.

The invention also provides a method for modifying the fatty acidprofile in oil, preferably increasing the oleic acid content, from aplant, preferably from oilseed rape, the method comprising the step ofintroducing a chimeric DNA into the cells of the plant, comprising thefollowing operably linked parts: a). a plant-expressible promoter,preferably a seed-specific promoter; b). a DNA region, particularly withthe nucleotide sequence of SEQ ID No 6, which when transcribed yields anRNA molecule comprising an RNA region capable of forming an artificialstem-loop structure, wherein one of the annealing RNA sequences of thestem-loop structure comprises a nucleotide sequence essentially similarto at least part of the nucleotide sequence of a Δ12 desaturase encodingopen reading frame, and wherein the second of the annealing RNAsequences comprises a sequence essentially similar to at least part ofthe complement of at least part of the nucleotide sequence of the Δ12desaturase encoding open reading frame; and optionally; c) a DNA regioninvolved in transcription termination and polyadenylation. Plants withmodified fatty acid profile, particularly with increased oleic acidcontent, comprising the mentioned chimeric genes are also provided bythe invention. Also encompassed are oils obtained from such plants orseed.

With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents schematically the different sense and antisenseconstructs, as well as the so-called CoP (complementary pair) constructsused for obtaining virus resistance (FIG. 1B) or for reducing thephenotypic expression of a transgenic Gus gene (FIG. 1A).

FIG. 2A represents schematically the so-called panhandle construct orCoP constructs used for reducing the phenotypic expression of a Δ12desaturase gene in Arabidopsis (Nos Pro: nopaline synthase genepromoter; nptII neomycin phospho-transferase coding region; Nos term:nopaline syntase gene terminator; FP1: truncated seed specific napinpromoter; 480 bp: 5′ end of the Fad2 gene of Arabidopsis thaliana insense orientation; 623 bp: spacer; 480 bp: 5′ end of the Fad2 gene ofArabidopsis thaliana in antisense orientation.

FIG. 2B represents schematically a common cosuppression construct forreducing the phenotypic expression of a Δ12 desaturase gene inArabidopsis thaliana.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

One of the objectives of the invention is to provide a eucaryotic cell,particularly a plant cell with an RNA molecule which comprises a hairpinstructure including a determined sense part and a determined antisensepart. Potentially, an RNA molecule is capable of forming severalsecondary structures, some of which may contain the desired hairpin. Itis expected that the real secondary structure of the RNA in the cell,will have the lowest free energy. In accordance with the invention, theRNA molecule to be produced in the cell is designed in such a way thatat least in its lowest free energy state, which it can assume withinthat cell, it will comprise the desired hairpin.

As used herein “hairpin RNA” refers to any self-annealing doublestranded RNA molecule. In its simplest representation, a hairpin RNAconsists of a double stranded stem made up by the annealing RNA strands,connected by a single stranded RNA loop, and is also referred to as a“pan-handle RNA”. However, the term “hairpin RNA” is also intended toencompass more complicated secondary RNA structures comprisingself-annealing double stranded RNA sequences, but also internal bulgesand loops. The specific secondary structure adapted will be determinedby the free energy of the RNA molecule, and can be predicted fordifferent situations using appropriate software such as FOLDRNA (Zukerand Stiegler, 1981).

As used herein, “sequence identity” with regard to nucleotide sequences(DNA or RNA), refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thetwo sequences. The alignment of the two nucleotide sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983)using a window-size of 20 nucleotides, a word length of 4 nucleotides,and a gap penalty of 4. Computer-assisted analysis and interpretation ofsequence data, including sequence alignment as described above, can,e.g., be conveniently performed using the programs of theIntelligenetics™ Suite (Intelligenetics Inc., CA). Sequences areindicated as “essentially similar” when such sequence have a sequenceidentity of at least about 75%, particularly at least about 80%, moreparticularly at least about 85%, quite particularly about 90%,especially about 95%, more especially about 100%, quite especially areidentical. It is clear than when RNA sequences are said to beessentially similar or have a certain degree of sequence identity withDNA sequences, thymine (T) in the DNA sequence is considered equal touracil (U) in the RNA sequence.

As used herein, the term “plant-expressible promoter” means a DNAsequence which is capable of controlling (initiating) transcription in aplant cell. This includes any promoter of plant origin, but also anypromoter of non-plant origin which is capable of directing transcriptionin a plant cell, i.e., certain promoters of viral or bacterial originsuch as the CaMV35S, the subterranean clover virus promoter No 4 or No7, or T-DNA gene promoters.

The term “expression of a gene” refers to the process wherein a DNAregion which is operably linked to appropriate regulatory regions,particularly to a promoter, is transcribed into an RNA which isbiologically active i.e., which is either capable of interaction withanother nucleic acid or which is capable of being translated into apolypeptide or protein. A gene is said to encode an RNA when the endproduct of the expression of the gene is biologically active RNA, suchas e.g. an antisense RNA, a ribozyme or a replicative intermediate. Agene is said to encode a protein when the end product of the expressionof the gene is a protein or polypeptide.

A nucleic acid of interest is “capable of being expressed”, when saidnucleic acid, when introduced in a suitable host cell, particularly in aplant cell, can be transcribed (or replicated) to yield an RNA, and/ortranslated to yield a polypeptide or protein in that host cell.

The term “gene” means any DNA fragment comprising a DNA region (the“transcribed DNA region”) that is transcribed into a RNA molecule (e.g.,an mRNA) in a cell operably linked to suitable regulatory regions, e.g.,a plant-expressible promoter. A gene may thus comprise several operablylinked DNA fragments such as a promoter, a 5′ leader sequence, a codingregion, and a 3′ region comprising a polyadenylation site. A plant geneendogenous to a particular plant species (endogenous plant gene) is agene which is naturally found in that plant species or which can beintroduced in that plant species by conventional breeding. A chimericgene is any gene which is not normally found in a plant species or,alternatively, any gene in which the promoter is not associated innature with part or all of the transcribed DNA region or with at leastone other regulatory region of the gene.

As used herein, “phenotypic expression of a nucleic acid of interest”refers to any quantitative trait associated with the molecularexpression of a nucleic acid in a host cell and may thus include thequantity of RNA molecules transcribed or replicated, the quantity ofpost-transcriptionally modified RNA molecules, the quantity oftranslated peptides or proteins, the activity of such peptides orproteins.

A “phenotypic trait” associated with the phenotypic expression of anucleic acid of interest refers to any quantitative or qualitativetrait, including the trait mentioned, as well as the direct or indirecteffect mediated upon the cell, or the organism containing that cell, bythe presence of the RNA molecules, peptide or protein, orposttranslationally modified peptide or protein. The mere presence of anucleic acid in a host cell, is not considered a phenotypic expressionor a phenotypic trait of that nucleic acid, even though it can bequantitatively or qualitatively traced. Examples of direct or indirecteffects mediated on cells or organisms are, e.g., agronomically orindustrial useful traits, such as resistance to a pest or disease;higher or modified oil content etc.

As used herein, “reduction of phenotypic expression” refers to thecomparison of the phenotypic expression of the nucleic acid of interestto the eucaryotic cell in the presence of the RNA or chimeric genes ofthe invention, to the phenotypic expression of the nucleic acid ofinterest in the absence of the RNA or chimeric genes of the invention.The phenotypic expression in the presence of the chimeric RNA of theinvention should thus be lower than the phenotypic expression in absencethereof, preferably be only about 25%, particularly only about 10%, moreparticularly only about 5% of the phenotypic expression in absence ofthe chimeric RNA, especially the phenotypic expression should becompletely inhibited for all practical purposes by the presence of thechimeric RNA or the chimeric gene encoding such an RNA.

A reduction of phenotypic expression of a nucleic acid where thephenotype is a qualitative trait means that in the presence of thechimeric RNA or gene of the invention, the phenotypic trait switches toa different discrete state when compared to a situation in which suchRNA or gene is absent. A reduction of phenotypic expression of a nucleicacid may thus, a.o., be measured as a reduction in transcription of(part of) that nucleic acid, a reduction in translation of (part of)that nucleic acid or a reduction in the effect the presence of thetranscribed RNA(s) or translated polypeptide(s) have on the eucaryoticcell or the organism, and will ultimately lead to altered phenotypictraits. It is clear that the reduction in phenotypic expression of anucleic acid of interest, may be accompanied by or correlated to anincrease in a phenotypic trait.

As used herein “a nucleic acid of interest” or a “target nucleic acid”refers to any particular RNA molecule or DNA sequence which may bepresent in a eucaryotic cell, particularly a plant cell.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA region which is functionallyor structurally defined, may comprise additional DNA regions etc.

It has unexpectedly been found by the inventors, that introduction of achimeric gene capable of being transcribed into an RNA molecule with anucleotide sequence comprising both the sense and antisense nucleotidesequence of a target gene, or part thereof, integrated in the nucleargenome of a plant cell, could efficiently and specifically reduce thephenotypic expression of that target gene. The reduction in phenotypicexpression was more efficient and more predictable than observed whenseparate chimeric genes were introduced in similar cells with the targetgene, encoding either sense or antisense RNA molecules.

At the same time, it has also been found that simultaneously introducingseparate chimeric genes in one cell encoding RNA molecules withnucleotide sequences comprising sense and antisense respectively,resulted in extreme virus resistance, even when the chimeric genes weretranscribed from weaker promoters. It is well known that gene silencingand virus resistance can be mediated by similar phenomena.

In one embodiment of the invention, a method for reducing the phenotypicexpression of a nucleic acid of interest, which is normally capable ofbeing expressed in a eucaryotic cell, particularly a plant cell, isprovided, comprising the steps of introducing a chimeric DNA comprisingthe following operably linked parts:

-   -   a) a promoter, operative in that cell, particularly a        plant-expressible promoter;    -   b) a DNA region capable of being transcribed into an RNA        molecule with a nucleotide sequence comprising        -   i. a sense nucleotide sequence of at least 10 nt, preferably            15 nt consecutive nucleotides having between 75 and 100%            sequence identity with at least part of the nucleotide            sequence of the nucleic acid of interest; and        -   ii. an antisense nucleotide sequence including at least 10,            preferably 15 nt consecutive nucleotides, having between            about 75% to about 100% sequence identity with the            complement of the at least 10, preferably 15 nt consecutive            nucleotides of the sense nucleotide sequence;    -   wherein the RNA is capable of forming a double stranded RNA by        base-pairing between the regions with sense and antisense        nucleotide sequence resulting in a hairpin RNA structure; and    -   c) a DNA region involved in transcription termination and        polyadenylation functioning in the suitable eucaryotic cells,        particularly functioning in plant cells.

In a preferred embodiment of the invention, the RNA molecule transcribedfrom the chimeric gene, consists essentially of the hairpin RNA.

The order of the sense and antisense nucleotide sequence in the RNAmolecule is thought not be critical.

Thus, in other words, the chimeric DNA has a transcribed DNA region,which when transcribed, yields an RNA molecule comprising an RNA regioncapable of forming an artificial stem-loop structure, wherein one of theannealing RNA sequences of the stem-loop structure comprises a sequence,essentially similar to at least part of the nucleotide sequence of thenucleic acid of interest, and wherein the second of the annealing RNAsequences comprises a sequence essentially similar to at least part ofthe complement of at least part of the nucleotide sequence of thenucleic acid of interest.

The RNA molecule may comprise several artificial hairpin structures,which may be designed to reduce the phenotypic expression of differentnucleic acids of interest.

In one preferred embodiment, the nucleic acid of interest, whosephenotypic expression is targeted to be reduced, is a gene incorporatedin the genome of a eucaryotic cell, particularly a plant cell. It willbe appreciated that the means and methods of the invention can be usedfor the reduction of phenotypic expression of a gene which belongs tothe genome of the cell as naturally occurring, (an endogenous gene), aswell as for the reduction of phenotypic expression of a gene which doesnot belong to the genome of the cell as naturally occurring, but hasbeen introduced in that cell (a transgene). The transgene can beintroduced stably or transiently, and can be integrated into the nucleargenome of the cell, or be present on a replicating vector, such as aviral vector.

In another preferred embodiment, the nucleic acid of interest, whosephenotypic expression is targeted to be reduced is a viral nucleic acid,particularly a viral RNA molecule, capable of infecting a eucaryoticcell, particularly a plant cell. In this case, the phenotype to bereduced is the replication of the virus, and ultimately, the diseasesymptoms caused by the infecting virus.

Preferably, the nucleotide sequence of the target nucleic acidcorresponding to the sense nucleotide sequence is part of a DNA regionwhich is transcribed, particularly a DNA region which is transcribed andtranslated (in other words a coding region). It is particularlypreferred that the target sequence corresponds to one or moreconsecutive exons, more particularly is located within a single exon ofa coding region.

The length of the sense nucleotide sequence may vary from about 10nucleotides (nt) up to a length equaling the length (in nucleotides) ofthe target nucleic acid. Preferably the total length of the sensenucleotide sequence is at least 10 nt, preferably 15 nt, particularly atleast about 50 nt, more particularly at least about 100 nt, especiallyat least about 150 nt, more especially at least about 200 nt, quiteespecially at least about 550 nt. It is expected that there is no upperlimit to the total length of the sense nucleotide sequence, other thanthe total length of the target nucleic acid. However for practicalreason (such as e.g. stability of the chimeric genes) it is expectedthat the length of the sense nucleotide sequence should not exceed 5000nt, particularly should not exceed 2500 nt and could be limited to about1000 nt.

It will be appreciated that the longer the total length of the sensenucleotide sequence is, the less stringent the requirements for sequenceidentity between the total sense nucleotide sequence and thecorresponding sequence in the target gene become. Preferably, the totalsense nucleotide sequence should have a sequence identity of at leastabout 75% with the corresponding target sequence, particularly at leastabout 80%, more particularly at least about 85%, quite particularlyabout 90%, especially about 95%, more especially about 100%, quiteespecially be identical to the corresponding part of the target nucleicacid. However, it is preferred that the sense nucleotide sequence alwaysincludes a sequence of about 10 consecutive nucleotides, particularlyabout 20 nt, more particularly about 50 nt, especially about 100 nt,quite especially about 150 nt with 100% sequence identity to thecorresponding part of the target nucleic acid. Preferably, forcalculating the sequence identity and designing the corresponding sensesequence, the number of gaps should be minimized, particularly for theshorter sense sequences.

The length of the antisense nucleotide sequence is largely determined bythe length of the sense nucleotide sequence, and will preferablycorrespond to the length of the latter sequence. However, it is possibleto use an antisense sequence which differs in length by about 10%.Similarly, the nucleotide sequence of the antisense region is largelydetermined by the nucleotide sequence of the sense region, andpreferably is identical to the complement of the nucleotide sequence ofthe sense region. Particularly with longer antisense regions, it ishowever possible to use antisense sequences with lower sequence identityto the complement of the sense nucleotide sequence, preferably with atleast about 75% sequence identity, more preferably with at least about80%, particularly with at least about 85%, more particularly with atleast about 90% sequence identity, especially with at least about 95%sequence to the complement of the sense nucleotide sequence.Nevertheless, it is preferred that the antisense nucleotide sequencesalways includes a sequence of about 10, preferably 15 consecutivenucleotides, particularly about 20 nt, more particularly about 50 nt,especially about 100 nt, quite especially about 150 nt with 100%sequence identity to the complement of a corresponding part of the sensenucleotide sequence. It is clear that the length of the stretch of theconsecutive nucleotides with 100% sequence identity to the complement ofthe sense nucleotide sequence cannot be longer than the sense nucleotidesequence itself. Again, preferably the number of gaps should beminimized, particularly for the shorter antisense sequences. Further, itis also preferred that the antisense sequence has between about 75% to100% sequence identity with the complement of the target sequence.

The RNA molecule resulting from the transcription of the chimeric DNAmay comprise a spacer nucleotide sequence located between the sense andantisense nucleotide sequence. In the absence of such a spacer sequence,the RNA molecule will still be able to form a double-stranded RNA,particularly if the sense and antisense nucleotide sequence are largerthan about 10 nucleotides and part of the sense and/or antisensenucleotide sequence will be used to form the loop allowing thebase-pairing between the regions with sense and antisense nucleotidesequence and formation of a double stranded RNA. It is expected thatthere are no length limits or sequence requirements associated with thespacer region, as long as these parameters do not interfere with thecapability of the RNA regions with the sense and antisense nucleotidesequence to form a double stranded RNA. In a preferred embodiment, thespacer region varies in length from 4 to about 200 bp, but as previouslymentioned, it may be absent.

In a preferred embodiment, the hairpin RNA formed by the sense andantisense region and if appropriate the spacer region, is an artificialhairpin RNA. By “artificial hairpin RNA” or “artificial stem-loop RNAstructure”, is meant that such hairpin RNA is not naturally occurring innature, because the sense and antisense regions as defined are notnaturally occurring simultaneously in one RNA molecule, or the sense andantisense regions are separated by a spacer region which is heterologouswith respect to the target gene, particularly, the nucleotide sequenceof the spacer has a sequence identity of less than 75% with thenucleotide sequence of the target sequence, at the correspondinglocation 5′ or 3′ of the endpoints of the sense nucleotide sequence. Ahairpin RNA can also be indicated as artificial, if it is not comprisedwithin the RNA molecule it is normally associated with. It isconceivable to use in accordance with the invention a chimeric DNA whosetranscription results in a hairpin RNA structure with a naturallyoccurring nucleotide sequence (which otherwise meets the limits as setforth in this specification) provided this hairpin RNA is devoid of thesurrounding RNA sequences (not involved in the hairpin structureformation).

Although it is preferred that the RNA molecule comprising the hairpinRNA does not further comprise an intron sequence, it is clear that thechimeric DNA genes encoding such RNAs may comprise in their transcribedregion one or more introns.

In fact, the inventors have unexpectedly found that inclusion of anintron sequence in the chimeric DNA genes encoding an RNA moleculecomprising the hairpin RNA, preferably in the spacer region, andpreferably in sense orientation, enhances the efficiency of reduction ofexpression of the target nucleic acid. The enhancement in efficiency maybe expressed as an increase in the frequency of plants wherein silencingoccurs or as an increase in the level of reduction of the phenotypictrait. In a particularly preferred embodiment, the intron is essentiallyidentical in sequence to the Flaveria trinervia pyruvate orthophosphatedikinase 2 intron 2, more particularly, it comprises the sequence of SEQID No 7.

It has been observed that contrary to methods using either antisense orsense nucleotide sequences alone to reduce the phenotypic expression ofa target nucleic acid (which generally depend on the dosage of sense orantisense molecule, and thus the chimeric genes encoding the sense andantisense molecules need to be under the control of relatively strongpromoters) the method of the current invention does not rely on thepresence of such strong promoter regions to drive the transcriptionalproduction of the RNA comprising both the sense and antisense region. Inother words, a whole range of promoters, particularly plant expressiblepromoters, is available to direct the transcription of the chimericgenes of the invention. These include, but are not limited to strongpromoters such as CaMV35S promoters (e.g., Harpster et al., 1988). Inthe light of the existence of variant forms of the CaMV35S promoter, asknown by the skilled artisan, the object of the invention can equally beachieved by employing these alternative CaMV35S promoters and variants.It is also clear that other plant-expressible promoters, particularlyconstitutive promoters, such as the opine synthase promoters of theAgrobacterium Ti- or Ri-plasmids, particularly a nopaline synthasepromoter, or subterranean clover virus promoters can be used to obtainsimilar effects. Also contemplated by the invention are chimeric genesto reduce the phenotypic expression of a nucleic acid in a cell, whichare under the control of single subunit bacteriophage RNA polymerasespecific promoters, such as a T7 or a T3 specific promoter, providedthat the host cells also comprise the corresponding RNA polymerase in anactive form.

It is a further object of the invention, to provide methods for reducingthe phenotypic expression of a nucleic acid in specific cells,particularly specific plant cells by placing the chimeric genes of theinvention under control of tissue-specific or organ-specific promoters.Such tissue-specific or organ-specific promoters are well known in theart and include but are not limited to seed-specific promoters (e.g.,WO89/03887), organ-primordia specific promoters (An et al., 1996),stem-specific promoters (Keller et al., 1988), leaf specific promoters(Hudspeth et al., 1989), mesophyl-specific promoters (such as thelight-inducible Rubisco promoters), root-specific promoters (Keller etal., 1989), tuber-specific promoters (Keil et al., 1989), vasculartissue specific promoters (Peleman et al., 1989), stamen-selectivepromoters (WO 89/10396, WO 92/13956), dehiscence zone specific promoters(WO 97/13865) and the like.

In another embodiment of the invention, the expression of a chimericgene to reduce the phenotypic expression of a target nucleic acid can becontrolled at will by the application of an appropriate chemicalinducer, by operably linking the transcribed DNA region of the chimericgenes of the invention to a promoter whose expression is induced by achemical compound, such as the promoter of the gene disclosed inEuropean Patent publication (“EP”) 0332104, or the promoter of the genedisclosed in WO 90/08826.

It has been found that a similar reduction in phenotypic expression of anucleic acid of interest in a eucaryotic cell, particularly in a plantcell, can be obtained by providing the sense and antisense RNA encodingtranscribable DNA regions as separate transgenes, preferably located inone locus, particularly as one allele.

Thus, in another embodiment of the invention a method for reducing thephenotypic expression of a nucleic acid which is normally capable ofbeing expressed in a eucaryotic cell, particularly a plant cell, isprovided, comprising the steps of introducing a first and secondchimeric DNA, linked on one recombinant DNA such that both chimeric DNAsare integrated together in the nuclear genome of the transgenic cells;

wherein the first chimeric DNA comprises the following operably linkedparts:

-   -   a) a promoter, operative in the cell, particularly a        plant-expressible promoter;    -   b) a DNA region capable of being transcribed into a sense RNA        molecule with a nucleotide sequence comprising a sense        nucleotide sequence of at least 10, preferably 15 consecutive        nucleotides having between 75 and 100% sequence identity with at        least part of the nucleotide sequence of the nucleic acid of        interest; and    -   c) a DNA region involved in transcription termination and        polyadenylation functioning in the corresponding eucaryotic        cell; and        wherein the second chimeric DNA comprises the following operably        linked parts:    -   a) a promoter, operative in the cell, particularly a        plant-expressible promoter;    -   b) a DNA region capable of being transcribed into an antisense        RNA molecule with a nucleotide sequence comprising an antisense        nucleotide sequence including at least 10, preferably 15        consecutive nucleotides, having between about 75% to about 100%        sequence identity with the complement of the at least 10,        preferably 15 consecutive nucleotides of the sense nucleotide        sequence;    -   c) a DNA region involved in transcription termination and        polyadenylation functioning in the corresponding eucaryotic        cell;        wherein the sense and antisense RNA are capable of forming a        double stranded RNA by base-pairing between the regions which        are complementary.

Preferred embodiments for the different structural and functionalcharacteristics, such as length and sequence of sense, antisense andspacer regions, of this method are as described elsewhere in thisspecification.

The RNA molecule, comprising the sense and antisense nucleotidesequences capable of forming a hairpin structure, which are produced bythe transcription of the chimeric genes, can also be introduced directlyin a plant cell to reduce the phenotypic expression of the targetnucleic acid, particularly to reduce the phenotypic expression of atargeted endogenous gene, or a targeted transgene. Such RNA moleculescould be produced e.g. by

-   1. cloning the DNA region capable of being transcribed into an RNA    molecule with a nucleotide sequence comprising a sense nucleotide    sequence of at least 10 consecutive nucleotides having between 75    and 100% sequence identity with at least part of the nucleotide    sequence of the nucleic acid of interest and an antisense nucleotide    sequence including at least 10 consecutive nucleotides, having    between about 75% to about 100% sequence identity with the    complement of the at least 10 consecutive nucleotides of the sense    nucleotide sequence, whereby the RNA is capable of forming a double    stranded RNA by base-pairing between the regions with sense and    antisense nucleotide sequence resulting in a hairpin RNA structure,    under control of a promoter suitable for recognition by a    DNA-dependent RNA polymerase in an in vitro transcription reaction,    such as but not limited to a T7-polymerase specific promoter;-   2. performing an in vitro transcription reaction by adding inter    alia the suitable DNA-dependent RNA polymerase as well as the    required reagents to generate the RNA molecules; and-   3. isolating the RNA molecules.

In vitro transcription methods as well as other methods for in vitro RNAproduction are well known in the art and commercial kits are available.Methods for direct introduction of RNA in plant cells are also availableto the skilled person and include but are not limited toelectroporation, microinjection and the like.

The chimeric gene(s) for reduction of the phenotypic expression of atarget nucleic acid of interest in a cell, may be introduced eithertransiently, or may be stably integrated in the nuclear genome of thecell. In one embodiment, the chimeric gene is located on a viral vector,which is capable of replicating in the eucaryotic cell, particularly theplant cell (see e.g., WO 95/34668 and WO 93/03161).

The recombinant DNA comprising the chimeric gene to reduce thephenotypic expression of a nucleic acid of interest in a host cell, maybe accompanied by a chimeric marker gene, particularly when the stableintegration of the transgene in the genome of the host cell isenvisioned. The chimeric marker gene can comprise a marker DNA that isoperably linked at its 5′ end to a promoter, functioning in the hostcell of interest, particularly a plant-expressible promoter, preferablya constitutive promoter, such as the CaMV 35S promoter, or a lightinducible promoter such as the promoter of the gene encoding the smallsubunit of Rubisco; and operably linked at its 3′ end to suitable planttranscription 3′ end formation and polyadenylation signals. It isexpected that the choice of the marker DNA is not critical, and anysuitable marker DNA can be used. For example, a marker DNA can encode aprotein that provides a distinguishable color to the transformed plantcell, such as the A1 gene (Meyer et al., 1987), can provide herbicideresistance to the transformed plant cell, such as the bar gene, encodingresistance to phosphinothricin (EP 0,242,246), or can provide antibioticresistance to the transformed cells, such as the aac(6′) gene, encodingresistance to gentamycin (WO94/01560).

A recombinant DNA comprising a chimeric gene to reduce the phenotypicexpression of a gene of interest, can be stably incorporated in thenuclear genome of a cell of a plant. Gene transfer can be carried outwith a vector that is a disarmed Ti-plasmid, comprising a chimeric geneof the invention, and carried by Agrobacterium. This transformation canbe carried out using the procedures described, for example, in EP 0 116718.

Alternatively, any type of vector can be used to transform the plantcell, applying methods such as direct gene transfer (as described, forexample, in EP 0 233 247), pollen-mediated transformation (as described,for example, in EP 0 270 356, WO85/01856 and U.S. Pat. No. 4,684,611),plant RNA virus-mediated transformation (as described, for example, inEP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediatedtransformation (as described, for example, in U.S. Pat. No. 4,536,475),and the like.

Other methods, such as microprojectile bombardment as described for cornby Fromm et al. (1990) and Gordon-Kamm et al. (1990), are suitable aswell. Cells of monocotyledonous plants, such as the major cereals, canalso be transformed using wounded and/or enzyme-degraded compactembryogenic tissue capable of forming compact embryogenic callus, orwounded and/or degraded immature embryos as described in WO92/09696. Theresulting transformed plant cell can then be used to regenerate atransformed plant in a conventional manner.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the chimeric gene for reduction of the phenotypicexpression of a nucleic acid of interest of the invention in othervarieties of the same or related plant species, or in hybrid plants.Seeds obtained from the transformed plants contain the chimeric genes ofthe invention as a stable genomic insert.

It is a further object of the invention to provide eucaryotic cells,preferably plant cells and organisms (preferably plants) comprising thechimeric genes for the reduction of the phenotypic expression of atarget nucleic acid as described in the invention.

It is a yet a further object of the invention to provide plant cells,comprising a nucleic acid of interest, which is normally capable ofbeing expressed phenotypically, further comprising an RNA molecule witha nucleotide sequence which includes:

-   i. a sense nucleotide sequence of at least 10 consecutive    nucleotides having between 75 and 100% sequence identity with at    least part of the nucleotide sequence of the nucleic acid of    interest; and-   ii. an antisense nucleotide sequence including at least 10    consecutive nucleotides, having between about 75% to about 100%    sequence identity with the complement of the at least 10 consecutive    nucleotides of the sense nucleotide sequence and capable of forming    a double stranded RNA by association with the sense nucleotide    sequence;    wherein the phenotypic expression of the nucleotide acid of interest    is significantly reduced by the presence of the RNA molecule, when    compared to the phenotypic expression of the nucleic acid of    interest in the absence of the RNA molecule. The RNA molecule may be    encoded by chimeric DNA. Preferred embodiments for the sense and    antisense nucleotide sequence, particularly concerning length and    sequence, are as mentioned elsewhere in this specification.

It will be appreciated that the methods and means described in thespecification can also be applied in High Throughput Screening (HTS)methods, for the identification or confirmation of phenotypes associatedwith the expression of a nucleic acid sequence with hithertounidentified function in a eucaryotic cell, particularly in a plantcell.

Such a method comprises the steps of1. selecting a target sequence within the nucleic acid sequence ofinterest with unidentified or non-confirmed function/phenotype whenexpressed. Preferably, if the nucleic acid has putative open readingframes, the target sequence should comprise at least part of one ofthese open reading frames. The length of the target nucleotide sequencemay vary from about 10 nucleotides up to a length equaling the length(in nucleotides) of the nucleic acid of interest with unidentifiedfunction.2. designing an RNA molecule comprising sense nucleotide sequence andantisense nucleotide sequence in accordance with the invention.3. introducing the RNA molecule comprising both the sense and antisensenucleotide sequences designed on the basis of the target sequence, intoa suited host cell, particularly a plant cell, comprising the nucleicacid with the nucleotide sequence with hitherto unidentified phenotype.The RNA can either be introduced directly, or can be introduced by meansof a chimeric DNA comprising a promoter operative in the host cell ofinterest, particularly a plant-expressible promoter, and a DNA regionfunctioning as a suitable 3′ end formation and polyadenylation signal(terminator) functioning in the host cell, with in-between a DNA regionwhich can be transcribed to yield the RNA molecule comprising the senseand antisense nucleotide sequence. The chimeric DNA can either beintroduced transiently or integrated in the nuclear genome. The chimericDNA can also be provided on a viral vector (see, e.g., WO 95/34668 andWO 93/03161)4. observing the phenotype by a suitable method. Depending on thephenotype expected, it may be sufficient to observe or measure thephenotype in a single cell, but it may also be required to culture thecells to obtain an (organized) multicellular level, or even toregenerate a transgenic organism, particularly a transgenic plant.

In its most straightforward embodiment, the RNA molecule comprising boththe sense and antisense nucleotide sequences to at least part of anucleic acid of interest, suitable for the methods of the invention, canbe obtained by cloning two copies of a DNA region with the selectedtarget sequence in inverted repeat orientation (preferably separated bya short DNA region which does not contain a transcription terminationsignal, and encodes the spacer sequence) under a suitable promoter. Thischimeric DNA is then either used as template DNA in an in vitrotranscription method to generate the RNA molecule, which is introducedin the host cell, or the chimeric DNA itself is introduced in the hostcell.

The methods and means of the invention can thus be used to reducephenotypic expression of a nucleic acid in a eucaryotic cell ororganism, particularly a plant cell or plant, for obtaining shatterresistance (WO 97/13865), for obtaining modified flower color patterns(EP 522 880, U.S. Pat. No. 5,231,020), for obtaining nematode resistantplants (WO 92/21757, WO 93/10251, WO 94/17194), for delaying fruitripening (WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO93/07275, WO 92/04456, U.S. Pat. No. 5,545,366), for obtaining malesterility (WO 94/29465, WO89/10396, WO 92/18625), for reducing thepresence of unwanted (secondary) metabolites in organisms, such asglucosinolates (WO97/16559) or chlorophyll content (EP 779 364) inplants, for modifying the profile of metabolites synthesized in aeucaryotic cell or organisms by metabolic engineering e.g. by reducingthe expression of particular genes involved in carbohydrate metabolism(WO 92/11375, WO 92/11376, U.S. Pat. No. 5,365,016, WO 95/07355) orlipid biosynthesis (WO 94/18337, U.S. Pat. No. 5,530,192), for delayingsenescence (WO 95/07993), for altering lignification in plants (WO93/05159, WO 93/05160), for altering the fibre quality in cotton (U.S.Pat. No. 5,597,718), for increasing bruising resistance in potatoes byreducing polyphenoloxidase (WO 94/03607), etc.

The methods of the invention will lead to better results and/or higherefficiencies when compared to the methods using conventional sense orantisense nucleotide sequences and it is believed that othersequence-specific mechanisms regulating the phenotypic expression oftarget nucleic acids might be involved and/or triggered by the presenceof the double-stranded RNA molecules described in this specification.

A particular application for reduction of the phenotypic expression of atransgene in a plant cell, inter alia, by antisense or sense methods,has been described for the restoration of male fertility, the latterbeing obtained by introduction of a transgene comprising a malesterility DNA (WO 94/09143, WO 91/02069). The nucleic acid of interestis specifically the male sterility DNA. Again, the processes andproducts described in this invention can be applied to these methods inorder to arrive at a more efficient restoration of male fertility.

The methods and means of the invention, particularly those involving RNAmolecules comprising a hairpin RNA and the encoding chimeric genes, haveproven to be particularly suited for the modification of the compositionof oil content in plants, particularly in seeds. Particularly preferredplants are crop plants used for oil production such as but not limitedto oilseed rape (Brassica juncea, napus, rapa, oleracea, campestris),corn, cotton, groundnut, sunflower, castor beans, flax, coconut,linseed, soybean. Preferred target genes are the desaturase genes,particularly Δ12 desaturase encoding genes such as those encoded by theFad2 genes, especially the genes whose nucleotide sequence can be foundin the Genbank Database under accession number AF123460 (from Brassicacarinata), AF12042841 (Brassica rapa), L26296 (Arabidopsis thaliana) orA65102 (Corylus avellana). It is clear that it is well within the reachof the person skilled in the art to obtain genes homologous to thedisclosed fad2 genes from other species e.g. by hybridization and/or PCRtechniques.

Preferred embodiments for the configuration of sense and antisensenucleotide sequences, particularly concerning length and sequence are asmentioned elsewhere in this specification. Also, preferred embodimentsfor chimeric genes encoding hairpin containing RNA molecules,particularly concerning promoter elements are as described elsewhere inthe specification. For this application, it is particularly preferredthat the promoters are seed-specific promoters.

In a preferred embodiment, the artificial hairpin RNA comprising RNAmolecule thus comprises part of a Δ12 desaturase encoding ORF in senseorientation and a similar part in antisense orientation, preferablyseparated by a spacer sequence. In a particularly preferred embodimentthe artificial hairpin RNA (or its encoding chimeric gene) comprises thenucleotide sequence of SEQ ID No 6 or a similarly constructed nucleotidesequence based on the aforementioned Brassica fad2 genes.

Preferably the chimeric gene encoding the artificial hairpin RNA istranscribed under control of a seed-specific promoter, particularlyunder control of the FPI promoter as described elsewhere in thisapplication.

A reduction of the expression of Δ12 desaturase gene in oil containingplants leads to increase in oleic acid and a concomitant decrease inlinolenic acid and linoleic acid. A higher frequency of plants with oilwherein the increase in oleic acid and concomitant decrease in linolenicand linoleic acid is significant is found using the means and methods ofthe invention than in transgenic plants harboring common cosuppressionconstructs. Moreover the absolute levels of increase, respectivelydecrease are higher respectively lower than in transgenic plantsharboring common cosuppression constructs.

Using the means and methods of the invention, it is thus possible toobtain plants and seeds, comprising an oil of which the compositionafter crushing and extracting has an increased oleic acid content(expressed as a percentage of the total fatty acid composition),particularly a three fold increase, when compared with control plants.

It is expected that using the methods and means of the invention,transgenic Brassica plant can be obtained, whose seeds comprise an oilwherein the oleic acid content exceeds 90% of the total fatty acidcontents.

The methods and means of the invention will be especially suited forobtaining virus resistance, particularly extreme virus resistance, ineucaryotic cells or organisms, particularly in plant cells and plants. Anon-limiting list of viruses for plants against which resistance can beobtained, is represented in Table I.

The methods and means of the invention further allow the use of viralgenes, hitherto unused for obtaining virus resistant plants in additionto the commonly used coat protein genes or replicase genes. Suchdifferent viral genes include protease encoded genes (Pro) genome linkedprotein (Vpg) encoding genes, cytoplasmic inclusion body encoding genes(CI) as target nucleic acid sequences for obtaining virus resistantplants.

It is clear that the invention will be especially suited for thereduction of phenotypic expression of genes belonging to multigenefamilies.

It is also clear that the methods and means of the invention are suitedfor the reduction of the phenotypic expression of a nucleic acid in allplant cells of all plants, whether they are monocotyledonous ordicotyledonous plants, particularly crop plants such as but not limitedto corn, rice, wheat, barley, sugarcane, cotton, oilseed rape, soybean,vegetables (including chicory, brassica vegetables, lettuce, tomato),tobacco, potato, and sugarbeet, but also plants used in horticulture,floriculture or forestry. The means and methods of the invention will beparticularly suited for plants which have complex genomes, such aspolyploid plants.

It is expected that the chimeric RNA molecules produced by transcriptionof the chimeric genes described herein, can spread systemicallythroughout a plant, and thus it is possible to reduce the phenotypicexpression of a nucleic acid in cells of a non-transgenic scion of aplant grafted onto a transgenic stock comprising the chimeric genes ofthe invention (or vice versa) a method which may be important inhorticulture, viticulture or in fruit production.

The following non-limiting Examples describe the construction ofchimeric genes for the reduction of the phenotypic expression of anucleic acid of interest in a eucaryotic cell and the use of such genes.Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA. Standard materials and methods for plant molecular workare described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK.

Throughout the description and Examples, reference is made to thefollowing sequences:

-   -   SEQ ID No 1: sequence of the Potato virus Y fragment of the Nia        gene used for the construction of various sense and antisense        constructs, for obtaining virus resistance.    -   SEQ ID No 2: sequence of the coding region of the Gusd CoP        construct of Example 1.    -   SEQ ID No 3: sequence of a modified 5′ untranslated region        (5′UTR) from Johnsongrass mosaic virus    -   SEQ ID No 4: sequence of the Subterranean clover virus promoter        No 4 with S7 enhancer.    -   SEQ ID No 5: sequence of the Subterranean clover virus double        enhancer promoter No 4.    -   SEQ ID No 6: sequence of the CoP construct for the Δ12        desaturase gene expression inhibition.    -   SEQ ID No 7: sequence of the Flaveria trinervia pyruvate        orthophosphate dikinase intron        The following free text is included in the sequence listing:

-   <223> fragment of the NIa ORF

-   <223> Description of Artificial Sequence:coding region of the Gusd    CoP construct

-   <223> deficient Gus coding region

-   <223> antisense to the 5′ end of the Gus coding region

-   <223> Description of Artificial Sequence:5′UTR of Johnson mosaic    virus

-   <223> Description of Artificial Sequence:Subterannean clover virus    S4 promoter with S7 enhancer

-   <223> Description of Artificial Sequence: subterranean clover virus    promoter S4 with S4 enhancer

-   <223> Description of Artificial Sequence: coding sequence of the    desaturase CoP construct

-   <223> corresponding to the 5′ end of the delta12-desaturase (fad2)    coding region, in sense orientation

-   <223> corresponding to the 5′ end of the delta12-desaturase (fad2)    coding region, in anti sense orientation

-   <223> Description of Artificial Sequence: intron 2 of the Flaveria    trinervia puryvate orthophosphate dikinase

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1 Experimental Procedures Gene Construction

Standard gene cloning methods (Sambrook et al. 1989) were used to makethe chimeric genes. A schematic representation of the constructs used isshown in FIGS. 1A and 1B.

The components for these constructs were:

Cauliflower mosaic virus 35S promoter from the Cabb-JI isolate(35S)(Harpster et al., 1988)Octopine synthase terminator (ocs-t) (MacDonald et al., 1991)Subterranean clover virus promoter No 4 (S4) (WO 9606932)Subterranean clover virus terminator No 4 (s4t) (WO 9606932)Subterranean clover virus double enhancer promoter No 4 (S4S4) (SEQ IDNo 5)Subterranean clover virus promoter No 4 with S7 enhancer (S7S4) (SEQ IDNo 4)Maize ubiquitin promoter (Ubi) (Christensen and Quail, 1996)Agrobacterium tumour morphology 1 gene terminator (tm1′) (Zheng et al.,1991)the Nia gene of an Australian strain of Potato virus Y (Nia) (SEQ ID No1)a dysfunctional 3-glucuronidase open reading frame encoding DNA(Gusd)(SEQ ID No 2 from nucleotide 1 to nucleotide 1581)a modified 5′ untranslated region (5′UTR) from Johnsongrass mosaic virus(JGMV5′) (SEQ ID No 3).This contains insertion of a NcoI site at the ATG start codon followedby three stop codons in frame, and a PstI site (for insertion of theintron as in constructs 4 and 5 of FIG. 1A). In vector constructs 2 and6 of FIG. 1A, the Gusd open reading frame is inserted in at the NcoIsite, removing the stop codons; in all other constructs of FIG. 1A it isinserted downstream of the PstI site.a castor bean catalase intron (Ohta et al., 1990) as modified by Wang etal. (1997) (“intron”).

The chimeric genes were constructed by operably assembling the differentparts as schematically indicated in FIG. 1A or FIG. 1B and inserting theobtained chimeric genes in the T-DNA vectors pART27 and pART7 vectors(Gleave, 1992) between the left T-DNA border and the chimeric plantexpressible neo gene.

The DNA encoding a dysfunctional β-glucuronidase open reading frame(GUSd) was obtained by deleting from a gus coding region the sequencebetween the two EcoRV restriction sites. For the construction of thechimeric gene encoding the RNA molecule comprising both sense andantisense nucleotide sequence to a β-glucuronidase gene, a sequence wasadded to the Gusd gene to be allow base pairing the 5′end over 558 basesresulting in a sequence as represented in SEQ ID No 2. This sequence wascloned between the maize ubiquitin promoter and the tm1′ terminator andinserted in a T-DNA vector.

T-DNA vectors were constructed which comprised a first and a secondchimeric virus resistance gene, wherein the first chimeric geneconsisted of:

-   -   1. a CaMV 35S promoter sequence, coupled to    -   2. in sense orientation, the nucleotide sequence from PVY        encoding either        -   Vpg protein (see e.g., Genbank Accession Nr ZZ9526 from            nucleotide 1013 to nucleotide 1583), or        -   part of the CI protein (see e.g., Genbank Accession Nr            M95491 from nucleotide 3688 to nucleotide 4215) or        -   Protease (Pro) (see e.g., EMBL Accession Nr D00441 from            nucleotide 5714 to nucleotide 7009), followed by    -   3. the S4 terminator from subterranean clover mosaic virus, as        described above.        The second chimeric gene consists of    -   1. a S4 promoter as described above, coupled to    -   2. in anti-sense orientation, the nucleotide sequence from PVY        encoding either        -   Vpg protein, or        -   CI protein or        -   Protease, followed by    -   3. the octopine synthase terminator as described above.        The sense and antisense sequences within one T-DNA vector were        derived from the same PVY coding region.

Also, T-DNA vectors were constructed for use in altering the fatty acidcomposition in oil (see FIG. 2), comprising

-   1. a FP1 promoter (truncated seed specific napin promoter,    containing sequences between −309 and +1, as described in Stalberg    et al; linked to-   2. the nucleotide sequence of SEQ ID No 6, comprising the 480 bp    located 5′ in the ORF encoding the Δ12 desaturase from Arabidopsis    thaliana (Fad2) in sense orientation and in antisense orientation,    linked by a 623 bp spacer sequence; followed by-   3. the terminator from the nopaline synthase gene.

In addition, T-DNA vectors were constructed to evaluate the influence ofa presence of an intron sequence in the chimeric genes encoding CoPconstructs. To this end, constructs were made comprising:

-   1. a CamV35S promoter, followed by-   2. the protease encoding ORF from PVY (see above) in sense    orientation;-   3. the sequence of SEQ ID No 7 (encoding the Flaveria trinervia    pyruvate orthophosphate dikinase intron 2)-   4. the protease encoding ORF from PVY in antisense orientation; and-   5. the octopine synthase gene terminator.

Plant Transformation

Nicotiana tabacum (W38) tissue was transformed and regenerated intowhole plants essentially as described by Landsman et al. (1988). Rice(Oryza sativa) was transformed essentially as described by Wang et al.(1997).

Rice Supertransformation

Mature embryos from a rice plant expressing GUS and hygromycinphosphotransferase (HPT) activity were excised from mature seed andplaced on callus inducing media for 7 weeks. Calli were recovered fromthese cultures, incubated with Agrobacteria containing various binaryvector constructs for 2 days, then placed on callusing media containinghygromycin, bialaphos and Timentin™. During the next four weekshygromycin and bialaphos resistant calli developed. These callus lineswere maintained on hygromycin and bialaphos containing media for afurther 2 months before being assayed for GUS activity.

GUS Assay

Rice calli were tested for GUS activity using the histochemical stainX-glucuronide or the fluorogenic substrate 4-methyl-umbeliferoneglucuronide (MUG) essentially as described by Jefferson et al. (1987).

Example 1. Comparison of Chimeric Genes Comprising Only Antisense, OnlySense, or Both Sense and Antisense (Complimentary Pair (CoP)) Sequencefor Reduction in Phenotypic Expression of an Integrated β-GlucuronidaseGene

Transgenic rice tissue expressing β-glucuronidase (GUS) from a singletransgene (and hygromycin resistance from a hph gene) (lines V10-28 andV10-67) was supertransformed using vectors that contained the bar geneconferring phosphinothricin resistance and various sense, antisense andCoP constructs (see FIG. 1A) derived from a crippled GUS (GUSd) gene.The supertransformed tissue was maintained on hygromycin and bialaphosselection media for 3 weeks then analyzed for GUS activity. A crippledGUS gene was used so that expression from this gene would not besuperimposed on the endogenous GUS activity.

The figures in Table 2 represent the rate of MU production measured byabsorption at 455 nm, with excitation at 365 nm of 1.5 μg of totalprotein in a reaction volume of 200 μl. The rate was measured over 30min at 37° C. The reading for non-transgenic rice calli was 0.162. Thefigures in bracket which follow the description of the introducedconstruct refer to FIG. 1A.

The results (Table 2) showed that supertransformation with the binaryvector containing the bar gene without the GUSd gene had no silencingeffect on the endogenous GUS activity. Supertransformation with GUSd ina sense or antisense orientation, with or without an intron or an earlystop codon, showed some degree of reduction (in about 25% of theanalyzed calli) of the endogenous GUS activity (see last two rows inTable 2 representing the percentage of analyzed calli with a MUG assayreading of less than 2.000). However, supertransformation with a CoPconstruct gave in about 75% to 100% of the analyzed calli, reduction ofthe endogenous GUS activity. This CoP construct was designed so that the3′ end of the mRNA produced could form a duplex with the 5′ end of thetranscript to give a “pan-handle” structure.

These data show that a complimentary pair can be made using oneself-annealing transcript, that this design is much more effective thana conventional sense or antisense construct, and that the approach canbe used to reduce the phenotypic expression of genes present in a plantcell.

TABLE 2 MUG assay of Supertransformed Rice Calli Anti- Vector Sense +sense + Inverted cassette Sense Sense + stop + stop + repeat (1) (2)Stop (3) intron (4) intron (5) CoP (6) V10-28 121.0 97.45 38.43 38.880.290 0.565 45.58 6.637 64.16 115.5 0.572 0.316 99.28 71.60 149.2 133.037.2 0.351 26.17 0.224 0.955 98.46 53.94 0.210 92.21 0.321 68.32 0.502105.5 0.701 108.8 5.290 105.6 39.35 56.73 0.733 6.432 0.9460 136.6 1.54560.36 2.103 90.80 32.44 140.4 10.36 71.12 119.8 98.24 128.8 62.38 111.613.17 0.717 93.76 31.28 17.79 14.42 0.424 0.398 5.023 88.06 26.98 0.31540.27 52.28 115.5 0.270 36.40 30.26 149.7 16.78 53.24 107.5 66.75 67.2829.97 26.75 145.8 0.217 89.06 105.1 0.534 0.208 0.256 135.1 9.4 68.2395.04 35.33 5.481 71.5 V10-67 318.8 93.43 0.199 31.82 1.395 0.472 109.573.19 0.197 58.08 152.4 0.256 30.35 128.1 0.157 56.32 67.42 0.296 40.041.506 128 44.62 12.11 0.452 228 140.6 130.3 0.454 0.668 0.422 23.051.275 196.2 17.32 23.34 0.196 241.2 0.272 12.43 73.2 76.10 0.294 118.50.209 140.0 20.32 130.1 0.172 11.27 42.05 90.13 107.4 0.841 0.436 110.6117.5 157.4 0.453 66.12 0.398 19.29 118.9 0.518 87.81 136.9 0.242 121.021.44 0.231 0.299 67.92 115.1 155.0 116.1 0.206 50.32 77.1 190.9 43.1812.47 170.3 106.1 0.773 31.06 0.213 108.9 73.12 0.146 11.15 1.241 29.9719.22 4.092 50.11 169.6 80.34 76.88 117.8 22.08 159.1 91.6 67.52 7.85592.32 69.76 27.97 0.822 V10-28 0%   21% 10% 10.5% 22%  75% V10-67 0%37.5% 33% 29.5% 21% 100%

Example 2. Comparison of the Efficiency of Using Chimeric GenesComprising Only Antisense Genes, Only Sense Genes, or Both GenesSimultaneously for Obtaining Virus Resistance in Transgenic Plants

Gene constructs were made using the PVY protease encoding sequence (SEQID No 1) in a sense orientation, an antisense orientation and in acomplimentary pair (CoP) orientation, where the T-DNA comprised both thesense and antisense chimeric genes each under control of their ownpromoter. In all three arrangements the CaMV35S promoter was used. Fivedifferent versions of CoP constructs were made in which the secondpromoter was either the CaMV35S promoter, the S4 promoter, the double S4promoter, the S7 enhanced S4 promoter, or the vascular specific rolCpromoter (see FIG. 1B).

These constructs were transformed into tobacco (via Agrobacteriummediated DNA transfer) and approximately 25 independently transformedplants were recovered per chimeric gene construct. The transgenic plantswere transferred to soil and maintained in the greenhouse. About 1 monthafter transplanting to soil, the plants were inoculated manually withpotato virus Y, using standard application methods. Two and four weekslater the plants were scored for virus symptoms. The results (Table 3)showed that after 1 month, 2 on 27 plants comprising only the sensegene, and 1 on 25 plants comprising only the antisense gene showed nosymptoms.

In contrast respectively 11 on 24 (35S-Nia/S4-antisenseNia construct), 7on 25 (35S-Nia/RolC-antisenseNia construct), 10 on 27(35S-Nia/35S-antisenseNia), 7 on 26 (35S-Nia/S4S4-antisenseNiaconstruct), and 7 on 25 (35S-Nia/S7S4-antisenseNia construct) plantswhich contained both the sense and antisense genes, showed no symptoms.Plants that showed no symptoms were considered to be showing extremeresistance to PVY. They continued to show no symptoms for a further 2months of monitoring (indicated as Extreme Resistant (ER) in Table 3).Some other plants, particularly those containing CoP constructs, showeda delay and restriction of symptoms. They showed no symptoms 2 weeksafter inoculation but showed some minor lesions in some plants after 4weeks. These plants were clearly much less effected by PVY thannon-transgenic or susceptible tobaccos and were scored as resistant(indicated as ER* in Table 3).

TABLE 3 Resistance to PVY infection of transgenic tobacco plantscomprising either the sense chimeric PVY protease construct, theantisense chimeric PVY protease construct, or both (different CoPconstructs). Extreme Total Resistant “Resistant” number of plants plantstransgenic Sense gene Antisense gene (ER) (ER*) plants 35S-Nia 2 2 2735S-AntisenseNia 1 0 25 35S-Nia 35S-AntisenseNia 10 2 27 35S-NiaS4-AntisenseNia 11 2 24 35S-Nia RolC-AntisenseNia 7 3 25 35S-NiaS4S4-AntisenseNia 7 7 26 35S-Nia S7S4-AntisenseNia 7 4 25The data show that using CoP constructs results in a much higherfrequency of transgenic plants with extreme resistance and resistancethan by using either sense or antisense constructs alone.

Next, the copy number of the transgenes in the virus resistanttransgenic plants was determined. Therefore, DNA was extracted from allthe transgenic plants showing extreme resistance or resistance. DNA wasalso extracted from five susceptible plants for each construct. The DNAwas examined for gene copy number using Southern analysis. The data(Table 4) showed that the genomes of some of the CoP plants showingextreme resistance, particularly the 35S-Nia/S4-AntisenseNia plants,only contained a single copy of the gene construct.

TABLE 4 Copy number of transgenes comprising sense chimeric PVY proteaseconstruct, the antisense chimeric PVY protease construct, or both(different CoP constructs) in extreme resistant, resistant andsusceptible plants. Extreme Sense Resistant “Resistant” Susceptible geneAntisense gene plants (ER) plants (ER*) plants 35S-Nia 6 1 1/1/1/1/135S-AntisenseNia 4 — 1/8/0/2/1 35S-Nia 35S-AntisenseNia 3/1/2/3/6/3/ 1/11/2/2/6/1 2/4/2/3 35S-Nia S4-AntisenseNia 2/4/1/3/2/4/ 2/6 5/8/2/36/1/1/3/1 35S-Nia RolC-AntisenseNia 6/7/6/7/7/7/6 2/1 2/2/2/1/2 35S-NiaS4S4-AntisenseNia 1/2/4/5/2/2/2 1/1/2/1/1/1/1 1/1/7/1/1 35S-NiaS7S4-AntisenseNia 2/4/12/5/2/2/ 3/2/1/2 1/1/3/1/1 7

Example 3. Inheritance of Extreme Resistance in Plants from Example 2

Plants from Example 2 were allowed to self-fertilize and their seedswere collected. Seeds originating from plants showing extreme resistanceand low transgene copy number for CoP constructs 35S-Nia/S4-AntisenseNiaand 35S-Nia/35S-AntisenseNia, and seeds from the sense and the antisenseplants showing extreme resistance, were germinated and grown in theglasshouse. Plants were also grown from seed collected from twosusceptible CoP lines, two susceptible sense gene only lines and twosusceptible antisense gene only lines. Twenty plants from each line wereselected for overall uniformity of size and development stage, put intoindividual pots, allowed to recover for one week, then inoculated withPVY. The plants were scored for virus symptoms 2, 4, and 7 weeks afterinoculation. The results (Table 5) showed that all eight plant lines of35S-Nia/S4-antisenseNia and 35S-Nia/35S-antisenseNia containing one ortwo gene copies showed an about 3:1 segregation ratio of extremeresistance:susceptible. The progeny of the single antisense gene onlyline that had given extreme resistance at T₀, and the progeny of theextremely resistant sense plant containing one gene copy, gave abnormalsegregation ratios (2:18; ER:susceptible). The progeny of the one senseplant that gave extreme resistance and contained 6 gene copies gave a˜3:1 ratio (ER:susceptible). All the progeny of the susceptible T₀plants showed complete susceptibility to PVY.

These data show extreme resistance from CoP constructs gives stableexpression of the resistance which is inherited in a Mendelian way. Thisalso indicates that, in these lines the PVY CoP gene loci are ˜100%effective at conferring extreme resistance whereas the transgene loci inthe antisense line and one of the two sense lines are only partiallyeffective at conferring extreme resistance.

TABLE 5 35S Sense 35S Sense 35S Nia and S4 Nia and S4 35S SenseAntisense Antisense Nia Antisense Nia Nia Nia Copy T1 Copy T1 Copy T1Copy T1 No ER:S No ER:S No ER:S No ER:S ER 1 15:5 1 16:4 6 17:3  4 2:18plant 1 2 1 12:8 2 15:5 1 2:18 3 1 14:6 2 16:4 4 1 15:5 2 16:4 Sus- 8 0:20 1  0:20 1 0:20 1 0:20 ceptible Plant 1 2 2  0:20 1  0:20 1 0:20 80:20

Example 4: Extreme Virus Resistant Transgenic Tobacco with DifferentComponents (Sense Gene and Antisense Gene) in Different Loci within theTransgenic Plant

PVY susceptible plants containing the sense transgene (which containedsingle transgene copies; see Table 6) were crossed with PVY susceptibleplants containing the antisense transgene (which had also been analyzedfor copy number by Southern analysis; see Table 6). Twenty of theresulting progeny per cross were propagated in the glasshouse, theninoculated with PVY and scored for virus infection as described inExample 2. The progeny from the crosses (between single genes/locicontaining plants) would be expected to be in the following ratio: ¼sense gene alone, ¼ antisense gene alone, ¼ comprising both sense andantisense genes, and ¼ comprising no genes at all. The results (Table 4)show that, with one exception, a proportion of the progeny from all thesuccessful crosses showed extreme resistance, whereas none of theprogeny from selfed sense or selfed antisense plants showed extremeresistance. The one cross that gave no extremely resistant progeny wasderived from the parent plant Antisense 2 (As2) which contained 8 copiesof the antisense gene. All twenty progeny plants from crosses Sense 1(S1)(male)×Antisense 1 (As1) (female) and Sense 3 (S3)(female)×Antisense 4 (As4) (male) were examined by Southern analysis.The results showed that in both crosses, the plants that showed extremeresistance (or in one case resistance) contained both the sense andantisense genes, whereas plants with no transgenes (nulls), or sense orantisense genes alone, were all susceptible to PVY. To further confirmthis absolute correlation between the presence of a complimentary pair(sense with antisense genes) within a plant and extreme resistance, allprogeny plants showing extreme resistance were analyzed by Southernblots. The results showed that every extremely resistant or resistantplant contained both sense and antisense genes.

These data show that a complimentary pair gives resistance or extremeresistance even when the genes encoding the sense and antisense genesare not co-located in the genome. The “complimentary pair phenomenon” isnot simply due to increased transgene dosage as it would be expectedthat ¼ of the selfed progeny would be homozygous and thus have doublethe gene dosage, yet they were susceptible.

TABLE 6 PVY resistance of the progeny plants resulting from crossesbetween susceptible transgenic tobacco plants comprising the35S-senseNia gene (S-lines) and susceptible transgenic tobacco plantscomprising the 35S-antisenseNia gene (As-lines). Extreme Resistant“Resistant” Male parent Female parent plants (ER) plants (ER*) S1 As1 8S1 As4 6 5 S2 As4 1 3 S3 As4 3 3 S4 As1 7 1 S3 As5 1 2 S4 As2 0 S4 As4 20 S5 As4 9 4 S5 As5 2 3 As4 As4 0 As5 As5 0 S2 S2 0 S4 S4 0Extreme resistant plants showed no symptoms of PVY infection after 7weeks. Resistant plants showed very minor lesions 7 weeks after PVYinfection. S1, S2, S3, S4 and S5 are PVY susceptible transgenic tobaccoplants comprising the 35S-senseNia gene construct which all have onecopy of the transgene integrated.

As1, As2, As4 and As5 are PVY susceptible transgenic tobacco plantscomprising the 35S-antisenseNia gene construct which have respectively1, 8, 2 and 1 copies of the transgene integrated.

Example 5. Evaluation of the Use of Different Viral Genes as TargetNucleic Acid Sequences in Obtaining Extreme Virus Resistant Genes

The T-DNA vectors comprising first and second chimeric virus resistancegenes based on sequences derived from the coding region for protease,Vpg or CI proteins from PVY as described in this application, were usedto obtain transformed tobacco plants, which were subsequently challengedwith PVY. The results are summarized in the following table:

TABLE 7 Number of immune plants/Number of independent transgenic plantsConstruct Replica 1 Replica 2 35S-Pro sense/S4-Pro antisense 11/24  7/2535S-Vpg sense/S4-Vpg antisense 8/20 6/18 35S-Cl sense/S4-Cl antisense2/23 1/20

Example 6. Intron Enhanced Silencing

The T-DNA vectors comprising the chimeric genes encoding the CoPconstructs wherein an intron (Flaveria trinervia pyruvate orthophosphatedikinase intron 2) has been inserted in either the sense orientation orthe antisense orientation, between the sense and antisense sequencescorresponding to the protease encoding ORF from PVY (as describedelsewhere in this application) were used to obtain transformed tobaccoplants, which were subsequently challenged with PVY. The results aresummarized in the following table:

TABLE 8 Number of immune plants/Number of Construct independenttransgenic plants 35S-Pro(sense)-intron(sense)- 22/24Pro(antisense)-Ocs-t 35S-Pro(sense)-intron(antisense)- 21/24Pro(antisense)-Ocs-t

Example 7. Modifying Oil Profile Using CoP Constructs in Arabidopsis

T-DNA vectors for modifying the fatty acid composition in oil, extractedfrom crushed seeds as described elsewhere in this application were usedto introduce the chimeric gene encoding the CoP construct for reducingthe expression (see FIG. 2A; SEQ ID No 6) the Δ12 desaturase gene (Fad2)in Arabidopsis thaliana.

For comparison of the efficiency, transgenic Arabidopsis plants weregenerated wherein the Fad2 gene expression was reduced by a plaincosuppression construct, comprising the FPI seed-specific promotercoupled to the complete ORF from the Δ12 desaturase gene (Fad2) inArabidopsis thaliana and the nopaline synthase promoter (see FIG. 2B).

As control plants, transgenic Arabidopsis transformed by unrelated T-DNAconstructs were used.

Seeds were harvested, crushed and extracted and the percentage of themajor fatty acids in the oil was determined by methods available in theart. The results, which are the mean of two readings, are summarized inTable 9.

TABLE 9 Peak Names Sample C18:1/ Name Myristic Palmitic PalmitoleicStearic Oleic Linoleic Linolenic 20:0 20:1 22:0 22:1 24:0 (C18:2 +C18:3) Hairpin 1.1 0.00 6.06 0.52 3.21 56.65 7.50 6.82 1.46 16.02 0.001.76 0.00 3.95 Hairpin 1.2 0.12 6.86 0.39 3.40 51.28 10.00 8.73 1.6415.60 0.00 1.97 0.00 2.74 Hairpin 1.3 0.11 8.47 0.50 3.49 21.64 28.9918.51 2.02 14.19 0.00 2.09 0.00 0.46 Hairpin 1.4 0.00 6.14 0.50 3.3751.70 9.77 8.02 1.73 16.04 0.00 2.05 0.67 2.91 Hairpin 2.1 0.06 5.190.43 3.33 54.84 5.52 7.76 1.77 18.50 0.34 1.83 0.45 4.13 Hairpin 2.20.04 7.67 0.46 3.75 19.60 28.29 18.64 2.55 15.96 0.19 2.28 0.56 0.42Hairpin 3.1 0.00 7.99 0.53 3.62 19.52 28.41 19.24 2.32 15.14 0.00 2.230.99 0.41 Hairpin 3.2 0.09 7.00 0.54 3.69 49.02 11.03 9.64 1.71 14.940.00 1.72 0.62 2.37 Hairpin 3.3 0.00 5.68 0.49 3.98 46.19 12.82 9.712.10 16.70 0.00 1.94 0.39 2.05 Hairpin 3.4 0.17 7.19 0.77 3.69 45.9011.86 10.65 1.84 15.39 0.00 1.90 0.65 2.04 Hairpin 3.5 0.00 6.45 0.483.26 51.76 8.13 10.04 1.51 16.08 0.00 1.92 0.36 2.85 Hairpin 3.6 0.087.51 0.23 3.59 19.97 29.13 20.12 2.15 14.54 0.29 2.02 0.36 0.41 Hairpin3.7 0.14 7.20 0.78 2.90 26.37 24.81 17.18 1.92 15.50 0.36 2.30 0.53 0.63Hairpin 3.8 0.11 6.34 0.46 3.23 38.58 15.25 13.54 1.89 16.91 0.00 2.361.34 1.34 Hairpin 3.9 0.00 6.47 0.49 3.32 47.59 11.44 9.63 1.68 15.960.00 1.88 1.55 2.26 Hairpin 3.10 0.00 6.77 0.56 3.48 53.30 7.57 9.341.55 15.65 0.00 1.79 0.00 3.15 Hairpin 3.11 0.00 7.05 0.59 3.61 53.628.87 8.36 1.55 14.35 0.00 1.99 0.00 3.11 Hairpin 3.12 0.05 8.32 0.363.85 18.48 29.24 19.94 2.48 14.75 0.00 2.28 0.26 0.38 Hairpin 4.1 0.096.97 0.59 3.61 53.64 8.40 8.44 1.60 15.00 0.00 1.66 0.00 3.19 Hairpin4.2 0.07 6.81 0.22 3.27 55.06 9.16 8.71 1.26 13.63 0.19 1.33 0.30 3.08Hairpin 4.3 0.04 6.81 0.50 3.47 46.21 10.67 11.52 1.81 16.50 0.00 1.880.58 2.08 Hairpin 5.1 0.00 8.30 0.23 3.71 17.72 28.92 20.63 2.38 14.770.00 2.41 0.92 0.36 Hairpin 5.2 0.19 7.15 1.55 3.56 44.58 11.44 11.591.77 15.67 0.00 1.84 0.65 1.94 Hairpin 5.3 0.10 6.49 0.40 3.72 54.197.01 7.89 1.74 15.91 0.00 1.92 0.62 3.64 Hairpin 5.5 0.12 6.58 0.51 3.8454.48 6.16 7.23 1.77 16.50 0.42 1.90 0.48 4.07 Hairpin 5.6 0.00 6.670.50 3.66 46.32 11.56 10.48 1.83 15.99 0.00 2.15 0.84 2.10 Hairpin 5.70.00 5.50 0.51 3.58 57.33 4.75 5.91 1.75 18.03 0.00 1.88 0.76 5.38Hairpin 5.8 0.16 6.55 1.53 3.54 48.52 9.91 8.97 1.78 16.39 0.00 1.840.81 2.57 Hairpin 6.1 0.10 6.35 0.57 3.48 59.00 4.77 6.26 1.48 15.950.00 1.80 0.25 5.35 Hairpin 6.2 0.10 7.98 0.37 4.06 20.96 29.01 18.692.38 13.63 0.20 2.03 0.60 0.44 Hairpin 6.5 0.08 6.21 0.63 3.61 60.055.07 5.27 1.55 15.20 0.00 1.69 0.66 5.81 Columbia pBin 0.08 8.81 0.473.51 17.07 30.31 20.94 1.78 14.56 0.00 2.17 0.28 0.33 19 controlCosuppresion 1.1 0.08 8.16 0.62 3.71 26.16 23.77 18.15 2.06 14.65 0.171.89 0.57 0.62 Cosuppresion 1.2 0.00 8.49 0.53 3.65 17.90 29.93 20.362.34 14.25 0.00 2.33 0.23 0.36 Cosuppresion 1.3 0.07 6.65 0.40 3.4238.34 15.25 14.16 1.91 17.19 0.31 1.94 0.35 1.30 Cosuppresion 1.4 0.008.22 0.57 3.82 18.27 28.82 19.63 2.56 14.83 0.00 2.46 0.83 0.38Cosuppresion 1.5 0.00 7.51 0.52 3.84 34.59 17.90 14.64 2.18 16.27 0.002.02 0.54 1.06 Cosuppresion 1.6 0.07 7.44 0.47 3.16 23.97 27.32 17.292.03 15.52 0.18 2.22 0.33 0.54 Cosuppresion 2.1 0.07 7.46 0.43 3.0023.91 27.21 17.79 1.84 15.27 0.30 2.14 0.58 0.53 Cosuppresion 2.2 0.008.19 0.55 4.22 18.59 28.31 18.80 2.77 15.51 0.00 2.46 0.58 0.39Cosuppresion 2.3 0.00 8.71 0.47 3.48 19.21 30.06 19.49 2.03 13.78 0.002.15 0.63 0.39 Cosuppresion 3.1 0.06 7.57 0.50 3.83 32.24 20.00 15.662.06 15.65 0.34 1.85 0.23 0.90 Cosuppresion 4.1 0.00 7.29 0.43 3.5530.26 21.17 17.06 2.01 16.08 0.00 1.92 0.25 0.79 Cosuppresion 4.2 0.088.02 0.53 3.62 33.04 20.04 15.68 1.80 14.72 0.00 1.88 0.58 0.92Cosuppresion 4.3 0.07 8.35 0.54 3.85 30.02 21.72 16.78 2.01 14.25 0.001.92 0.49 0.78 Cosuppresion 4.4 0.06 6.98 0.53 3.62 43.38 13.24 12.771.74 15.37 0.30 1.67 0.33 1.67 Cosuppresion 4.5 0.13 7.84 0.52 3.7633.76 18.16 16.21 1.89 14.96 0.35 1.85 0.57 0.98 Cosuppresion 4.6 0.118.18 0.32 3.58 19.72 29.19 20.26 2.04 13.92 0.29 1.84 0.55 0.40Cosuppresion 4.7 0.11 7.88 0.39 3.75 27.40 22.85 17.44 2.08 15.29 0.002.04 0.76 0.68 Cosuppresion 4.8 0.13 7.56 0.41 3.46 32.27 20.50 15.451.90 15.47 0.00 2.02 0.83 0.90 Cosuppresion 4.9 0.09 7.46 0.29 3.7536.11 16.96 15.74 1.92 15.38 0.31 1.74 0.25 1.10 Cosuppresion 5.1 0.107.68 0.34 3.88 36.00 16.77 15.38 1.90 15.44 0.32 1.82 0.36 1.12Cosuppresion 5.2 0.08 7.56 0.25 3.58 26.10 25.11 17.79 1.96 15.03 0.301.72 0.54 0.61 Cosuppresion 5.3 0.08 7.38 0.20 3.56 42.24 13.33 13.321.76 15.19 0.16 1.61 1.18 1.59 Cosuppresion 6.1 0.08 8.04 0.50 3.6831.37 20.29 17.17 1.84 14.31 0.00 1.76 0.95 0.84 Cosuppresion 6.2 0.008.50 0.51 3.91 18.59 29.33 19.66 2.46 14.75 0.00 2.28 0.00 0.38 Controlc24 0.07 8.30 0.10 4.78 19.68 25.91 20.56 2.97 15.29 0.31 1.79 0.24 0.42pGNAP-p450

Analysis of the results indicates that transgenic plants harboring a CoPconstruct (indicated as “hairpin x.x” in the table) have a higherfrequency of plants with oil wherein the increase in oleic acid andconcomitant decrease in linolenic and linoleic acid is significant thanin transgenic plants harboring cosuppression constructs. Moreover theabsolute levels of increase, respectively decrease are higherrespectively lower than in transgenic plants harboring cosuppressionconstructs.

Example 7. Modifying Oil Profile Using CoP Constructs in Brassica

The T-DNA vector harboring the chimeric gene encoding the CoP constructdescribed in Example 6 is introduced in Brassica oilseed rape. Seedsharvested from the transgenic Brassica sp. are crashed and oil extractedand the composition of the fatty acids in the oil is analyzed.

Oil from transgenic Brassica sp. harboring the CoP construct havesignificantly increased oleic acid content and decreased linoleic andlinolenic acid content. A T-DNA vector harboring a chimeric geneencoding a CoP construct similar to the one described in Example 6, butwherein the sequence of the sense and antisense region corresponding tothe Δ12 desaturase encoding ORF is based on a homologous ORF fromBrassica spp. is constructed and introduced in Brassica oilseed rape.

The sequence of Brassica spp ORFs homologous to Δ12 desaturase encodingORF from Arabidopsis are available from Genbank database under Accessionnrs AF042841 and AF124360.

Seeds harvested from the transgenic Brassica sp. are crashed and oilextracted and the composition of the fatty acids in the oil is analyzed.Oil from transgenic Brassica sp. harbouring the CoP construct havesignificantly increased oleic acid content and decreased linoleic andlinolenic acid content.

Example 8. Suppression of an Endogenous Rust Resistance Gene in Flax

A CoP construct for suppression of the endogenous rust resistance genewas made consisting of

1. a CaMV35S promoter; operably linked to

2. part of an endogenous rust resistance gene (n) from flax (about 1500bp long) in the sense orientation; ligated to

3. a similar part of the endogenous rust resistance gene from flax(about 1450 bp long) in antisense orientation so that a perfect invertedrepeat without spacer sequence is generated wherein each repeat is about1450 bp long; followed by

4. a nos terminator.

Plain antisense constructs were made using a similar fragment asdescribed sub 3 above inserted between a CaMV35S promoter and a nosterminator.

Flax plants containing the n gene (which gives resistance to a strain offlax rust) were transformed by these CoP and antisense constructs. Ifsuppression occurs, the plants become susceptible to the rust strain. Ifthe construct has no effect, the transformed plants remain resistant tothe rust strain.

Results

ngc-b sense/antisense 3 suppressed out of 7 ngc-b antisense 0 suppressedout of 12

While the invention has been described and illustrated herein byreferences to various specific material, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial, combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied and will beappreciated by those skilled in the art.

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1. (canceled)
 2. A method for reducing the phenotypic expression of anucleic acid of interest, which is normally capable of being expressedin a plant cell, comprising the step of introducing a chimeric DNAcomprising the following operably linked parts: a) a promoter, operativein the plant cell; b) a DNA region, which when transcribed, yields anRNA molecule comprising an RNA region capable of forming an artificialhairpin RNA structure comprising two annealing RNA sequences wherein oneof the annealing RNA sequences of the hairpin RNA structure comprises asense sequence that is identical to at least 100 consecutive nucleotidesof a nucleotide sequence of the nucleic acid of interest and wherein thesecond of said annealing RNA sequences comprises an antisense sequencewith is identical to at least 100 consecutive nucleotides of thecomplement of at least part of the nucleotide sequence of the nucleicacid of interest and wherein the sense and antisense sequences of theartificial hairpin structure are not naturally occurring in one RNAmolecule or the sense and antisense sequences are separated by a spacerregion with is heterologous with respect to the target gene; and c) aDNA region involved in transcription termination and polyadenylation.3-12. (canceled)
 13. A method for reducing the phenotypic expression ofa nucleic acid of interest, which is normally capable of being expressedin a plant cell comprising the step of introducing into the plant cell achimeric RNA molecule comprising at least one RNA region with anucleotide sequence comprising i. a sense nucleotide sequence of atleast 15 consecutive nucleotides having 100% sequence identity with atleast part of the nucleotide sequence of the nucleic acid of interest;and ii. an antisense nucleotide sequence including at least 15consecutive nucleotides, having about 100% sequence identity with thecomplement of said at least 15 consecutive nucleotides of said sensenucleotide sequence; wherein said RNA region is capable of forming anartificial hairpin RNA structure with a double stranded RNA stem bybase-pairing between the sense and antisense nucleotide sequences suchthat said at least 15 consecutive nucleotides of the sense sequencebasepair with said at least 15 consecutive nucleotides of the antisensesequence. 14-20. (canceled)
 21. A method for obtaining a phenotypeassociated with the reduction of expression of a nucleic acid ofinterest in a plant cell, said method comprising a. selecting withinsaid nucleotide sequence of interest, a target sequence of at least 15consecutive nucleotides; b. designing a sense nucleotide sequencecorresponding to the length of the selected target sequence and whichhas a sequence identity of about 100% with said selected targetsequence; c. designing an antisense nucleotide sequence which: i) has asequence identity of about 100% with the complement of said at least 15consecutive nucleotides of said sense nucleotide sequence; ii) comprisesa stretch of at least about 15 consecutive nucleotides with 100%sequence identity to the complement of a part of said sense nucleotidesequence; d. introducing an RNA molecule comprising both said sense andantisense nucleotide sequences into a plant cell comprising the nucleicacid of interest; and e. observing the phenotype by a suitable method.22. (canceled)
 23. A plant cell, comprising a nucleic acid of interest,which is normally capable of being phenotypically expressed, furthercomprising a chimeric RNA molecule comprising at least one RNA regionwith a nucleotide sequence comprising i. a sense nucleotide sequence ofat least 15 consecutive nucleotides having 100% sequence identity withat least part of the nucleotide sequence of the nucleic acid ofinterest; and ii. an antisense nucleotide sequence including at least 15consecutive nucleotides, having about 100% sequence identity with thecomplement of said at least 15 consecutive nucleotides of said sensenucleotide sequence; wherein said RNA is capable of forming anartificial hairpin RNA with a double stranded RNA region by base-pairingbetween the sense and antisense nucleotide sequences such that said atleast 15 consecutive nucleotides of the sense sequence basepair withsaid at least 15 consecutive nucleotides of the antisense sequence.24-25. (canceled)
 26. A plant comprising the plant cell of claim 23.27-38. (canceled)
 39. The method of claim 13, wherein said RNA moleculefurther comprises a spacer nucleotide sequence located between saidsense and said antisense nucleotide sequences.
 40. The method of claim39, wherein said spacer nucleotide sequence has a length between 4 and200 nucleotides.
 41. The method of claim 13, wherein said nucleic acidof interest is a gene integrated in the genome of said plant cell. 42.The method of claim 41, wherein said gene is an endogenous gene.
 43. Themethod of claim 41, wherein said gene is a foreign transgene.
 44. Themethod of claim 13, wherein said nucleic acid of interest is comprisedin the genome of an infecting virus.
 45. The method of claim 44, whereinsaid infecting virus is an RNA virus.
 46. The method of claim 13,wherein said plant cell is comprised within a plant.
 47. The method ofclaim 13, wherein said sense nucleotide sequence includes at least 20consecutive nucleotides having between 95% and 100% sequence identitywith at least 20 consecutive nucleotides of said part of the nucleotidesequence of said nucleic acid of interest, and said antisense nucleotidesequence includes at least 20 consecutive nucleotides having between 95%and 100% sequence identity with the complement of said at least 20consecutive nucleotides of said sense nucleotide sequence.
 48. Themethod of claim 13, wherein said sense nucleotide sequence includes atleast 50 consecutive nucleotides having between 95% and 100% sequenceidentity with at least 50 consecutive nucleotides of said part of thenucleotide sequence of said nucleic acid of interest, and said antisensenucleotide sequence includes at least 50 consecutive nucleotides havingbetween 95% and 100% sequence identity with the complement of said atleast 50 consecutive nucleotides of said sense nucleotide sequence. 49.The method of claim 13, wherein said sense nucleotide sequence includesat least 100 consecutive nucleotides having between 95% and 100%sequence identity with at least 100 consecutive nucleotides of said partof the nucleotide sequence of said nucleic acid of interest, and saidantisense nucleotide sequence includes at least 100 consecutivenucleotides having between 95% and 100% sequence identity with thecomplement of said at least 100 consecutive nucleotides of said sensenucleotide sequence.
 50. The method of claim 13, wherein said RNAmolecule is generated by transcription from a template DNA whichcomprises two copies of a DNA region, each copy comprising the sensenucleotide sequence, and the two copies being in an inverted repeatorientation and being under control of a promoter.
 51. The method ofclaim 50, wherein said transcription is an in vitro transcriptionmethod.
 52. The method of claim 21, wherein said RNA molecule isgenerated by transcription from a template DNA which comprises twocopies of a DNA region, each copy comprising the selected targetsequence, the two copies being in an inverted repeat orientation andbeing under control of a promoter.
 53. The method of claim 52, whereinsaid transcription is an in vitro transcription method.
 54. The methodof claim 13, wherein said introducing of the chimeric RNA molecule intosaid plant cell is liposome-mediated.
 55. The method of claim 21,wherein said introducing of the RNA molecule into said plant cell isliposome-mediated.
 56. The plant cell of claim 23, wherein said RNAmolecule further comprises a spacer nucleotide sequence located betweensaid sense and said antisense nucleotide sequences.
 57. The plant cellof claim 56, wherein said spacer nucleotide sequence has a lengthbetween 4 and 200 nucleotides.
 58. The plant cell of claim 23, whereinsaid sense nucleotide sequence includes at least 20 consecutivenucleotides having between 95% and 100% sequence identity with at least20 consecutive nucleotides of said part of the nucleotide sequence ofsaid nucleic acid of interest, and said antisense nucleotide sequenceincludes at least 20 consecutive nucleotides having between 95% and 100%sequence identity with the complement of said at least 20 consecutivenucleotides of said sense nucleotide sequence.
 59. The plant cell ofclaim 23, wherein said sense nucleotide sequence includes at least 50consecutive nucleotides having between 95% and 100% sequence identitywith at least 50 consecutive nucleotides of said part of the nucleotidesequence of said nucleic acid of interest, and said antisense nucleotidesequence includes at least 50 consecutive nucleotides having between 95%and 100% sequence identity with the complement of said at least 50consecutive nucleotides of said sense nucleotide sequence.
 60. The plantcell of claim 23, wherein said sense nucleotide sequence includes atleast 100 consecutive nucleotides having between 95% and 100% sequenceidentity with at least 100 consecutive nucleotides of said part of thenucleotide sequence of said nucleic acid of interest, and said antisensenucleotide sequence includes at least 100 consecutive nucleotides havingbetween 95% and 100% sequence identity with the complement of said atleast 100 consecutive nucleotides of said sense nucleotide sequence. 61.The plant cell of claim 23, wherein said nucleic acid of interest is agene integrated in the genome of said plant cell.
 62. The plant cell ofclaim 61, wherein said gene is an endogenous gene.
 63. The plant cell ofclaim 62, wherein said gene is a foreign transgene.
 64. The plant cellof claim 23, wherein said nucleic acid of interest is comprised in thegenome of an infecting virus.
 65. The plant cell of claim 64, whereinsaid infecting virus is an RNA virus.
 66. The plant cell of claim 23,wherein said RNA molecule is generated by transcription from a templateDNA which comprises two copies of a DNA region, each copy comprising thesense nucleotide sequence, and the two copies being in an invertedrepeat orientation and being under control of a promoter.
 67. The plantcell of claim 66, wherein said transcription is an in vitrotranscription method.